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  • 201.
    Gräslund, Susanne
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Eklund, Malin
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Falk, Ronny
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Nygen, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, nr 1, s. 41-50Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

  • 202.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Ehn, Maria
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Gunnel, Lundin
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, nr 1-2, s. 157-166Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

  • 203.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 93-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 204.
    Gräslund, Torbjörn
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hober, Sophia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 93-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 205.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Lundin, Gunnel
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Charge engineering of a protein domain to allow efficient ion-exchange recovery2000Ingår i: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, nr 10, s. 703-709Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

  • 206.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nilsson, Joakim
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Lindberg, Michael
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein1997Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, s. 125-132Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

  • 207. Gulich, S.
    et al.
    Linhult, M.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Stability towards alkaline conditions can be engineered into a protein ligand2000Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, nr 2, s. 169-178Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.

  • 208. Gulich, S.
    et al.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography2000Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, nr 03-feb, s. 233-244Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.

  • 209. Gummesson, A.
    et al.
    Björnson, E.
    Fagerberg, Linn
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zhong, Wen
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Abdellah, Tebani
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Edfors, Fredrik
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Schmidt, C.
    Lundqvist, A.
    Adiels, M.
    Bäckhed, F.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Jansson, P. -A
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Bergström, G.
    Longitudinal plasma protein profiling of newly diagnosed type 2 diabetes2021Ingår i: EBioMedicine, E-ISSN 2352-3964, Vol. 63, artikel-id 103147Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Comprehensive proteomics profiling may offer new insights into the dysregulated metabolic milieu of type 2 diabetes, and in the future, serve as a useful tool for personalized medicine. This calls for a better understanding of circulating protein patterns at the early stage of type 2 diabetes as well as the dynamics of protein patterns during changes in metabolic status. Methods: To elucidate the systemic alterations in early-stage diabetes and to investigate the effects on the proteome during metabolic improvement, we measured 974 circulating proteins in 52 newly diagnosed, treatment-naïve type 2 diabetes subjects at baseline and after 1 and 3 months of guideline-based diabetes treatment, while comparing their protein profiles to that of 94 subjects without diabetes. Findings: Early stage type 2 diabetes was associated with distinct protein patterns, reflecting key metabolic syndrome features including insulin resistance, adiposity, hyperglycemia and liver steatosis. The protein profiles at baseline were attenuated during guideline-based diabetes treatment and several plasma proteins associated with metformin medication independently of metabolic variables, such as circulating EPCAM. Interpretation: The results advance our knowledge about the biochemical manifestations of type 2 diabetes and suggest that comprehensive protein profiling may serve as a useful tool for metabolic phenotyping and for elucidating the biological effects of diabetes treatments. Funding: This work was supported by the Swedish Heart and Lung Foundation, the Swedish Research Council, the Erling Persson Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish state under the agreement between the Swedish government and the county councils (ALF-agreement).

  • 210. Gustafsson, A. C.
    et al.
    Kijas, J. M. H.
    Alderborn, A.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Andersson, L.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing2001Ingår i: Animal Biotechnology, ISSN 1049-5398, E-ISSN 1532-2378, Vol. 12, nr 2, s. 145-153Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The increasing interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) emphasis the need for high-throughput and cost effective scoring methods. Pyrosequencing is a novel method for screening SNPs. In this study we examine breed specific SNPs in the pig melanocortin I receptor gene (MC1R), some causing coat color phenotypes. A total of fifteen pigs representing eight breeds and crosses were analyzed by pyrosequencing. In addition to nine previously known SNPs, we also detected one new missense mutation by pyrosequencing. We here show that the SNPs were readily scored using standard reaction conditions. Insertions as well as substitutions were unambiguously detected and all genotypes were resolved in terms of homo- and heterozygozity.

  • 211. Gustavsson, Elin
    et al.
    Ek, Sara
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kristensson, Malin
    Älgenäs, Cajsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wingren, Christer
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Borrebaeck, Carl A. K.
    Surrogate antigens as targets for proteome-wide binder selection2011Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 28, nr 4, s. 302-311Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

  • 212.
    Gyllensten, Ulf
    et al.
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden.;Stellenbosch Inst Adv Study STIAS, Marais Rd, ZA-7600 Stellenbosch, South Africa..
    Hedlund-Lindberg, Julia
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden..
    Svensson, Johanna
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Manninen, Johanna
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Ost, Torbjorn
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Ramsell, Jon
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Aslin, Matilda
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Ivansson, Emma
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden..
    Lomnytska, Marta
    Uppsala Univ, Dept Womens & Childrens Hlth, SE-75185 Uppsala, Sweden..
    Lycke, Maria
    Gothenburg Univ, Inst Clin Sci, Dept Obstet & Gynaecol, Sahlgrenska Acad, SE-41685 Gothenburg, Sweden..
    Axelsson, Tomas
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Liljedahl, Ulrika
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Nordlund, Jessica
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Edqvist, Per-Henrik
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden..
    Sjoblom, Tobias
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH Royal Inst Technol, Sci Life Lab, SE-17165 Stockholm, Sweden..
    Stalberg, Karin
    Uppsala Univ, Dept Womens & Childrens Hlth, SE-75185 Uppsala, Sweden..
    Sundfeldt, Karin
    Gothenburg Univ, Inst Clin Sci, Dept Obstet & Gynaecol, Sahlgrenska Acad, SE-41685 Gothenburg, Sweden..
    Aberg, Mikael
    Uppsala Univ, Dept Med Sci, SE-75237 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, SE-75237 Uppsala, Sweden..
    Enroth, Stefan
    Uppsala Univ, Biomed Ctr, Dept Immunol Genet & Pathol, SciLifeLab Uppsala, SE-75108 Uppsala, Sweden.;Swedish Coll Adv Study, Thunbergsvagen 2, SE-75238 Uppsala, Sweden..
    Next Generation Plasma Proteomics Identifies High-Precision Biomarker Candidates for Ovarian Cancer2022Ingår i: Cancers, ISSN 2072-6694, Vol. 14, nr 7, s. 1757-, artikel-id 1757Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Simple Summary Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. The aim of our study was to broadly measure protein biomarkers to find tests for the early detection of ovarian cancer. We found that combinations of 4-7 protein biomarkers can provide highly accurate detection of early- and late-stage ovarian cancer compared to benign conditions. The performance of the tests was then validated in a second independent cohort. Background: Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. Methods: We employed the Explore PEA technology for high-precision analysis of 1463 plasma proteins and conducted a discovery and replication study using two clinical cohorts of previously untreated patients with benign or malignant ovarian tumours (N = 111 and N = 37). Results: The discovery analysis identified 32 proteins that had significantly higher levels in malignant cases as compared to benign diagnoses, and for 28 of these, the association was replicated in the second cohort. Multivariate modelling identified three highly accurate models based on 4 to 7 proteins each for separating benign tumours from early-stage and/or late-stage ovarian cancers, all with AUCs above 0.96 in the replication cohort. We also developed a model for separating the early-stage from the late-stage achieving an AUC of 0.81 in the replication cohort. These models were based on eleven proteins in total (ALPP, CXCL8, DPY30, IL6, IL12, KRT19, PAEP, TSPAN1, SIGLEC5, VTCN1, and WFDC2), notably without MUCIN-16. The majority of the associated proteins have been connected to ovarian cancer but not identified as potential biomarkers. Conclusions: The results show the ability of using high-precision proteomics for the identification of novel plasma protein biomarker candidates for the early detection of ovarian cancer.

  • 213. Haab, B. B.
    et al.
    Paulovich, A. G.
    Leigh Anderson, N.
    Clark, A. M.
    Downing, G. J.
    Hermajakob, H.
    LaBaer, J.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A reagent resource to identify proteins and peptides of interest for the cancer community2006Ingår i: Molecular and Cellular Proteomics, ISSN 1535-9476, Vol. 5, nr 10, s. 1996-2007Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    On the basis of discussions with representatives from all sectors of the cancer research community, the National Cancer Institute (NCI) recognizes the immense opportunities to apply proteomics technologies to further cancer research. Validated and well characterized affinity capture reagents (e.g. antibodies, aptamers, and affibodies) will play a key role in proteomics research platforms for the prevention, early detection, treatment, and monitoring of cancer. To discuss ways to develop new resources and optimize current opportunities in this area, the NCI convened the "Proteomic Technologies Reagents Resource Workshop" in Chicago, IL on December 12-13, 2005. The workshop brought together leading scientists in proteomics research to discuss model systems for evaluating and delivering resources for reagents to support MS and affinity capture platforms. Speakers discussed issues and identified action items related to an overall vision for and proposed models for a shared proteomics reagents resource, applications of affinity capture methods in cancer research, quality control and validation of affinity capture reagents, considerations for target selection, and construction of a reagents database. The meeting also featured presentations and discussion from leading private sector investigators on state-of-the-art technologies and capabilities to meet the user community's needs. This workshop was developed as a component of the NCI's Clinical Proteomics Technologies Initiative for Cancer, a coordinated initiative that includes the establishment of reagent resources for the scientific community. This workshop report explores various approaches to develop a framework that will most effectively fulfill the needs of the NCI and the cancer research community.

  • 214. Habuka, Masato
    et al.
    Fagerberg, Linn
    Hallstrom, Bjorn M.
    Ponten, Fredrik
    Yamamoto, Tadashi
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    The Urinary Bladder Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling2015Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 10, nr 12, artikel-id e0145301Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To understand functions and diseases of urinary bladder, it is important to define its molecular constituents and their roles in urinary bladder biology. Here, we performed genome-wide deep RNA sequencing analysis of human urinary bladder samples and identified genes upregulated in the urinary bladder by comparing the transcriptome data to those of all other major human tissue types. 90 protein-coding genes were elevated in the urinary bladder, either with enhanced expression uniquely in the urinary bladder or elevated expression together with at least one other tissue (group enriched). We further examined the localization of these proteins by immunohistochemistry and tissue microarrays and 20 of these 90 proteins were localized to the whole urothelium with a majority not yet described in the context of the urinary bladder. Four additional proteins were found specifically in the umbrella cells (Uroplakin 1a, 2, 3a, and 3b), and three in the intermediate/basal cells (KRT17, PCP4L1 and ATP1A4). 61 of the 90 elevated genes have not been previously described in the context of urinary bladder and the corresponding proteins are interesting targets for more in-depth studies. In summary, an integrated omics approach using transcriptomics and antibody-based profiling has been used to define a comprehensive list of proteins elevated in the urinary bladder.

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  • 215.
    Habuka, Masato
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska University Hospital, Sweden .
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kampf, Caroline
    Edlund, Karolina
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Yamamoto, Tadashi
    Pontén, Fredrik
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Odeberg, Jacob
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska University Hospital, Sweden.
    The Kidney Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling2014Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 9, nr 12, s. e116125-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into tissue enriched, group enriched or tissue enhanced. Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n=11), proximal tubules (n=120), distal tubules (n=9) or collecting ducts (n=8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease.

  • 216. Hamsten, C.
    et al.
    Skattum, L.
    Truedsson, L.
    von Döbeln, U.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hammarström, L.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Heat differentiated complement factor profiling2015Ingår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 126, s. 155-162Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

  • 217.
    Hamsten, Carl
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Westberg, Joakim
    Bölske, Göran
    Ayling, Roger
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.2008Ingår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, s. 539-549Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

  • 218. Hamsten, Carl
    et al.
    Wiklundh, Emil
    Gronlund, Hans
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlen, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Eklund, Anders
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Grunewald, Johan
    Haggmark-Manberg, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Elevated levels of FN1 and CCL2 in bronchoalveolar lavage fluid from sarcoidosis patients2016Ingår i: Respiratory Research, ISSN 1465-9921, E-ISSN 1465-993X, Vol. 17, artikel-id 69Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Sarcoidosis is a granulomatous systemic inflammatory disease in which more than 90 % of all patients develop pulmonary manifestations. Several gene associations have previously been described, but established and clinically useful biomarkers are still absent. This study aimed to find proteins in bronchoalveolar lavage (BAL) fluid that can be associated with the disease. Methods: We developed and performed profiling of 94 selected proteins in BAL fluid and serum samples obtained from newly diagnosed and non-treated patients with sarcoidosis. Using multiplexed immunoassays, a total of 317 BAL and 217 serum samples were analyzed, including asthmatic patients and healthy individuals as controls. Results: Our analyses revealed increased levels of eight proteins in sarcoidosis patients compared to controls. Out of these, fibronectin (FN1) and C-C motif chemokine 2 (CCL2) revealed the strongest associations. In addition, cadherin 5 (CDH5) was found to correlate positively with lymphocyte cell numbers in BAL fluid. Conclusions: Applying a high throughput proteomics screening technique, we found proteins of potential clinical relevance in the context of sarcoidosis.

  • 219. Han, L.
    et al.
    Wei, X.
    Liu, C.
    Volpe, G.
    Zhuang, Z.
    Zou, X.
    Wang, Z.
    Pan, T.
    Yuan, Y.
    Zhang, X.
    Fan, P.
    Guo, P.
    Lai, Y.
    Lei, Y.
    Liu, X.
    Yu, F.
    Shangguan, S.
    Lai, G.
    Deng, Q.
    Liu, Y.
    Wu, L.
    Shi, Q.
    Yu, H.
    Huang, Y.
    Cheng, M.
    Xu, J.
    Wang, M.
    Wang, C.
    Zhang, Y.
    Xie, D.
    Yang, Y.
    Yu, Y.
    Zheng, H.
    Wei, Y.
    Huang, F.
    Lei, J.
    Huang, W.
    Zhu, Z.
    Lu, H.
    Wang, B.
    Chen, F.
    Yang, T.
    Du, W.
    Chen, J.
    Xu, S.
    An, J.
    Ward, C.
    Pei, Z.
    Wong, C. -W
    Zhang, H.
    Liu, M.
    Qin, B.
    Schambach, A.
    Isern, J.
    Feng, L.
    Guo, X.
    Liu, Z.
    Sun, Q.
    Maxwell, P. H.
    Barker, N.
    Muñoz-Cánoves, P.
    Gu, Y.
    Mulder, Jan
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Tan, T.
    Liu, S.
    Yang, H.
    Wang, J.
    Hou, Y.
    Xu, X.
    Esteban, M. A.
    Liu, L.
    Cell transcriptomic atlas of the non-human primate Macaca fascicularis2022Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 604, nr 7907, s. 723-731Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell–cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs. 

  • 220. Hau, Sofie Olsson
    et al.
    Karnevi, Emelie
    Lundgren, Sebastian
    Li, Bo
    Lynch, Seodhna
    Elebro, Jacob
    Heby, Margareta
    Nodin, Bjorn
    Eberhard, Jakob
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Moran, Bruce
    Gallagher, William M.
    Jirstrom, Karin
    A translational effort to identify prognostic and predictive biomarkers in pancreatic cancer among RBM3-regulated genes.2018Ingår i: Journal of Clinical Oncology, ISSN 0732-183X, E-ISSN 1527-7755, Vol. 36, nr 4Artikel i tidskrift (Övrigt vetenskapligt)
  • 221. Hedberg, Jesper
    et al.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Donnes, Pierre
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Molecular profiling of human kidney injury using antibody suspension bead arrays2009Ingår i: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 189, s. S94-S94Artikel i tidskrift (Övrigt vetenskapligt)
  • 222.
    Hedhammar, My
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Gräslund, Torbjörn
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Negatively charged purification tags for selective anion-exchange recovery2004Ingår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, nr 11, s. 779-786Artikel i tidskrift (Refereegranskat)
    Abstract [en]

     A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

  • 223.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lönneborg, Rosa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nord, Olof
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjölin, Olle
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Uhlén, Matthias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein2005Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, nr 2, s. 133-146Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

  • 224. Hedner, Charlotta
    et al.
    Gaber, Alexander
    Korkocic, Dejan
    Nodin, Bjorn
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kuteeva, Eugenia
    Johannesson, Henrik
    Jirstrom, Karin
    Eberhard, Jakob
    SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma2014Ingår i: Virchows Archiv, ISSN 0945-6317, E-ISSN 1432-2307, Vol. 465, nr 6, s. 649-659Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype.

  • 225. Hertzberg, M.
    et al.
    Aspeborg, H.
    Schrader, J.
    Andersson, A.
    Erlandsson, R.
    Blomqvist, K.
    Bhalerao, R.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Sundberg, B.
    Nilsson, Peter
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Sandberg, G.
    A transcriptional roadmap to wood formation2001Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 98, nr 25, s. 14732-14737Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

  • 226.
    Hikmet, Feria
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, Uppsala, Sweden..
    Mear, Loren
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, Uppsala, Sweden..
    Edvinsson, Asa
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, Uppsala, Sweden..
    Micke, Patrick
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. Karolinska Inst, Dept Neurosci, Stockholm, Sweden..
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, Uppsala, Sweden..
    The protein expression profile of ACE2 in human tissues2020Ingår i: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 16, nr 7, artikel-id e9610Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The novel SARS-coronavirus 2 (SARS-CoV-2) poses a global challenge on healthcare and society. For understanding the susceptibility for SARS-CoV-2 infection, the cell type-specific expression of the host cell surface receptor is necessary. The key protein suggested to be involved in host cell entry is angiotensin I converting enzyme 2 (ACE2). Here, we report the expression pattern of ACE2 across > 150 different cell types corresponding to all major human tissues and organs based on stringent immunohistochemical analysis. The results were compared with several datasets both on the mRNA and protein level. ACE2 expression was mainly observed in enterocytes, renal tubules, gallbladder, cardiomyocytes, male reproductive cells, placental trophoblasts, ductal cells, eye, and vasculature. In the respiratory system, the expression was limited, with no or only low expression in a subset of cells in a few individuals, observed by one antibody only. Our data constitute an important resource for further studies on SARS-CoV-2 host cell entry, in order to understand the biology of the disease and to aid in the development of effective treatments to the viral infection.

  • 227. Hjalmarsson, S
    et al.
    Alderborn, A
    Fock, C
    Muldin, I
    Kling, H
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Engstrand, L
    Rapid combined characterization of microorganism and host genotypes using a single technology2004Ingår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 9, nr 2, s. 138-145Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background. Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrose-quencing(TM) technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. Materials and Methods. DNA from 87 clinical isolates of H. pylori 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. Results. All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. Conclusions. We conclude that genetic analysis using Pyrosequencing(TM) technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markets.

  • 228.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Brennan, Donal J
    Zendehrokh, Nooreldin
    Eberhard, Jakob
    Nodin, Björn
    Gaber, Alexander
    Pontén, Fredrik
    Johannesson, Henrik
    Smaragdi, Kristina
    Frantz, Christian
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Johnson, Louis B
    Påhlman, Sven
    Jirström, Karin
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer2011Ingår i: Proteomics. Clinical applications, ISSN 1862-8354, Vol. 5, nr 11-12, s. 624-35Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Purpose: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. Experimental design: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). Results: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. Conclusion and clinical relevance: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.

  • 229.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Ingår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 230.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Igel, Ulrika
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, Henrik
    Stadler, Charlotte
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Sjoberg, Anna
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Christine
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Generation of monospecific antibodies based on affinity capture of polyclonal antibodies2011Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 11, s. 1824-1835Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

  • 231.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 232.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes2012Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 233.
    Hober, Andreas
    et al.
    KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.
    Edfors, Fredrik
    KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.
    Ryaboshapkina, Maria
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Malmqvist, Jonas
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Rosengren, Louise
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Percy, Andrew J.
    Cambridge Isotope Labs Inc, Dept Applicat Dev, Tewksbury, MA 01876 USA.
    Lind, Lars
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk epidemiologi.
    Forsström, Björn
    KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.
    Uhlen, Mathias
    KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.
    Oscarsson, Jan
    AstraZeneca, Global Med Dev Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Miliotis, Tasso
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay2019Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, nr 12, s. 2433-2446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

    Ladda ner fulltext (pdf)
    fulltext
  • 234.
    Hober, Andreas
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Edfors, Fredrik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ryaboshapkina, Maria
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Malmqvist, Jonas
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Rosengren, Louise
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Percy, Andrew J.
    Cambridge Isotope Labs Inc, Dept Applicat Dev, Tewksbury, MA 01876 USA..
    Lind, Lars
    Uppsala Univ, Dept Med Sci, Uppsala, Sweden..
    Forsström, Björn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Oscarsson, Jan
    AstraZeneca, Global Med Dev Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Miliotis, Tasso
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay2019Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, nr 12, s. 2433-2446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

  • 235.
    Hober, Andreas
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hua, Tranh-Min Khue
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Foley, Dominic
    Waters Corp, Milford, England..
    McDonald, Thomas
    Waters Corp, Milford, England..
    Vissers, Johannes Pc
    Waters Corp, Milford, England..
    Pattison, Rebecca
    Waters Corp, Milford, England..
    Ferries, Samantha
    Waters Corp, Milford, England..
    Hermansson, Sigurd
    Waters Corp, Milford, England..
    Betner, Ingvar
    Waters Corp, Milford, England..
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Razavi, Morteza
    SISCAPA Assay Technol Inc, Victoria, BC, Canada..
    Yip, Richard
    SISCAPA Assay Technol Inc, Victoria, BC, Canada..
    Pope, Matthew E.
    SISCAPA Assay Technol Inc, Victoria, BC, Canada..
    Pearson, Terry W.
    SISCAPA Assay Technol Inc, Victoria, BC, Canada..
    Andersson, Leigh N.
    SISCAPA Assay Technol Inc, Victoria, BC, Canada..
    Bartlett, Amy
    Waters Corp, Milford, England..
    Calton, Lisa
    Waters Corp, Milford, England..
    Alm, Jessica J.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Solna, Sweden.;Karolinska Inst, Natl Pandem Ctr, Solna, Sweden..
    Engstrand, Lars
    Karolinska Inst, Microbiol Tumour & Cell Biol, Stockholm, Sweden..
    Edfors, Fredrik
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis2021Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 10, artikel-id e70843Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct <= 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

  • 236.
    Hober, Andreas
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rekanovic, Mirela
    AstraZeneca, Gothenburg, Sweden.
    Forsström, Björn
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Hansson, Sara
    AstraZeneca, Gothenburg, Sweden.
    Kotol, David
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Percy, Andrew J
    Cambridge Isotope Laboratories, Inc..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Oscarsson, Jan
    AstraZeneca, Gothenburg, Sweden.
    Edfors, Fredrik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Miliotis, Tasso
    AstraZeneca, Gothenburg, Sweden.
    Targeted Proteomics Using Stable Isotope Labeled Protein Fragments Enables Precise and Robust Determination of Total Apolipoprotein(a) in Human PlasmaManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on proteotypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.7%) and inter-assay repeatability (CV: 7.8%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a kringle 4 specific peptide allows for the determination of molar concentration and relative size of apo(a) in individuals.

    Ladda ner (pdf)
    abstract
  • 237.
    Hober, Andreas
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Rekanovic, Mirela
    Translational Science and Experimental Medicine, Cardiovascular, Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Forsström, Björn
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Hansson, Sara
    Translational Science and Experimental Medicine, Cardiovascular, Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Kotol, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Percy, Andrew J.
    Department of Applications Development, Cambridge Isotope Laboratories, Inc., Tewksbury, Massachusetts, United States of America.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Oscarsson, Jan
    Late-stage Development, Cardiovascular, Renal and Metabolism, Biopharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Edfors, Fredrik
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Miliotis, Tasso
    Translational Science and Experimental Medicine, Cardiovascular, Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Targeted proteomics using stable isotope labeled protein fragments enables precise and robust determination of total apolipoprotein(a) in human plasma2023Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 18, nr 2 February, artikel-id e0281772Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/ MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on proteotypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.7%) and inter-assay repeatability (CV: 7.8%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a kringle 4 specific peptide allow for the determination of molar concentration and relative size of apo(a) in individuals.

  • 238.
    Hober, Andreas
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Strandberg, Linnéa
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    von Feilitzen, Kalle
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Kotol, David
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Forsström, Björn
    Atlas Antibodies AB, Bromma, Sweden.
    Sjöberg, Anna
    Atlas Antibodies AB, Bromma, Sweden.
    Tegel, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Edfors, Fredrik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Evaluation of an enhanced antibody-validation strategy for Western blot applications based on migration pattern recognitionManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The use of affinity reagents, such as antibodies, for studying specific molecules in complex backgrounds are some of the most powerful tools for researchers in molecular biology. However, all experiments performed using affinity reagents are directly affected by each reagent’s context-dependent ability to bind specifically to a target of interest. A growing issue with non-validated, or poorly validated affinity reagents, has been highlighted by the International Working Group for Antibody Validation (IWGAV). It has been suggested that antibodies should be evaluated in an application-specific manner since they can perform well in one application but fail to deliver reproducible results in another. One of the most commonly used antibody-based applications is the Western blot (WB) technology. When evaluating the result from a WB experiment, the initial measure used for determining whether or not the antibody binds the protein of interest is to determine the molecular weight of the protein detected by the antibody compared to a set of reference proteins. As WB relies on the SDS-PAGE for separating differently sized proteins, the comparison is actually based on protein migration during electrophoresis. It is, however, well known that the migration of a protein can differ significantly from how the reference proteins migrate. Here, we suggest a method for determining the actual migration patterns of proteins instead of relying on the theoretical molecular weight of the protein. Using this approach, called migration capture mass spectrometry (MS), a dataset containing the migration patterns of more than 39,000 protein products from more than 10,500 genes across eleven cell lines and tissues has been created. This migration capture MS approach has been validated using k-fold cross validation against 249 siRNA knockdown WBs showing that the method has a sensitivity of 96.4%, specificity of 87.4% and accuracy of 91.9%, which makes the dataset a useful resource that can facilitate antibody validation strategies in a fit-for-purpose manner. The data set has allowed the automatic evaluation of more than 12,000 antibodies in the Human Protein Atlas using the method.

    Ladda ner (pdf)
    abstract
  • 239.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Hansson, A
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nilsson, B
    Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.1994Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 33, nr 22Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.

  • 240.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Lundström Ljung, J
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nilsson, B
    Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions.1999Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 443, nr 3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.

  • 241.
    Hober, Sophia
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Human protein atlas and the use of microarray technologies2008Ingår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 19, nr 1, s. 30-35Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies.

  • 242. Holgersson, G.
    et al.
    Ekman, S.
    Reizenstein, J.
    Bergqvist, M.
    Pontén, F.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Magnusson, K.
    Jonnalagadda, P.
    Asplund, A.
    Strömberg, S.
    Linder, A.
    Blomquist, E.
    Liljeholm, M.
    Löden, B.
    Hellström, K.
    Bergström, S.
    Molecular profiling using tissue microarrays as a tool to identify predictive biomarkers in laryngeal cancer treated with radiotherapy2010Ingår i: Cancer Genomics & Proteomics, ISSN 1109-6535, E-ISSN 1790-6245, Vol. 7, nr 1, s. 1-7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aim: To explore the usefulness of the expression of five potential cancer biomarkers in predicting outcome in patients with laryngeal cancer. Materials and Methods: In the present study, the Swedish National Cancer Registry databases were used to identify patients with laryngeal cancer diagnosed during the years 1978-2004 in the Uppsala-Örebro region and treated with radiotherapy. The expression of Ki-67, MutS homolog 2, (MSH2), p53, B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1 in the cancer cells was assessed immunohistochemically using tissue microarrays (TMAs) and its predictve value on survival and relapse was analyzed using Cox regression models. Results: A total of 39 patients were included in the present study. Nuclear MSH2 staining was statistically significantly correlated to Ki-67 expression (p=0.022). However, univariate and multivariate Cox analyses showed no statistically significant association between the expression of the investigated biomarkers and overall survival or relapse. Conclusion: The present exploratory study does not show any significant predictive value of the biomarkers examined with respect to survival or relapse. However, with larger patient cohorts, we believe that protein profiling using TMAs and immunohistochemistry is a feasible strategy for prognostic and predictive biomarker screening in laryngeal cancer.

  • 243. Holmberg, A.
    et al.
    Blomstergren, A.
    Nord, O.
    Lukacs, M.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures2005Ingår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, nr 3, s. 501-510Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K-d, in the order of 4 x 10(-14) m. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70degreesC can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluiclics and nanotechnology.

  • 244.
    Holmberg, A.
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Fry, G.
    Applied Biosystems, 850 Lincoln Center Drive, Foster City, 94404, CA, United States.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Automatic Preparation of DNA Templates for Sequencing on the ABI Catalyst Robotic Workstation1994Ingår i: Automated DNA Sequencing and Analysis / [ed] M.D. Adams, C. Fields, J.C. Venter, Elsevier Ltd , 1994, s. 139-145Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    The need for automation of the various steps in DNA sequencing increases as the number of applications and genomic sequencing applications grow. It is therefore necessary to design robotic workstations for template preparation and sequencing reactions in order to allow high throughput of samples with reproducible results of high quality. Also, it is not economically feasible to use manual labor in such large-scale projects.

    Several protocols based on paramagnetic microbeads for the recovery and purification of DNA templates to be used for sequencing have recently been described. These protocols involve both purification of polymerase chain reaction (PCR) products (Hultman et al., 1989) and M13 templates (Fry et al., 1992) and allow the isolation of specific single-stranded DNA templates. Protocols using both T7 DNA polymerase (Tabor & Richardson, 1989) and Taq cycle sequencing (Carothers et al., 1989) have been investigated. The fact that pure single-stranded templates are obtained allows accurate sequencing of heterozygotes using T7 DNA polymerase (Gibbs et al., 1989; Syvänen et al. 1991), mixed viral populations such as human immunodeficiency virus (HIV) (Wahlberg et al., 1992a) and direct mitochondrial sequencing of hair shafts and semen (Hopgood et al., 1992). The advantages of using magnetic beads as solid support include the possibility to perform manipulations such as strand melting and hybridization in a small volume with rapid kinetics (Uhlén, 1989). It is also important to point out that the magnetic beads can be transferred with a simple pipette tool, thus allowing immobilized DNA to be aliquoted and mixed within different wells or tubes within a robotic workstation. Thus, easy liquid handling is combined with rapid phase separation using a magnetic field. Recently, several automated protocols for solid phase sequencing have been described based on the Beckman Biomek-1000 laboratory workstation (Hultman et al., 1991; Wahlberg et al., 1992b).

    Here, we describe an experimental model of a robotic workstation (ABI Catalyst) to allow for magnetic preparation of PCR products and/or M13 templates to be used directly for sequencing either with T7 DNA polymerase or Taq DNA polymerase. A novel user interface to allow for modifications of the protocols is also described. Thus, automatic protocols with flexible parameter settings are obtained, allowing an easy setup of integrated protocols for template preparations and sequencing reactions.

  • 245.
    Hong, Mun-Gwan
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Dodig-Crnkovic, Tea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Chen, Xu
    Drobin, Kimi
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lee, Woojoo
    Wang, Yunzhang
    Edfors, Fredrik
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Kotol, David
    Thomas, Cecilia Engel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sjöberg, Ronald
    Odeberg, Jacob
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Cellulär och klinisk proteomik.
    Hamsten, Anders
    Silveira, Angela
    Hall, Per
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Pawitan, Yudi
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Pedersen, Nancy L
    Hägg, Sara
    Magnusson, Patrik KE
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality2020Ingår i: Life Science Alliance, E-ISSN 2575-1077, Vol. 3, nr 10, s. e202000817-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50–92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10−6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12–1.39; P = 6.45 × 10−5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7–9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.

  • 246. Hot, B.
    et al.
    Valnohova, J.
    Arthofer, E.
    Simon, K.
    Shin, J.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kostenis, E.
    Mulder, J.
    Schulte, G.
    FZD10-Gα13 signalling axis points to a role of FZD10 in CNS angiogenesis2017Ingår i: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 32, s. 93-103Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both β-catenin-dependent and –independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12 Gαi1 GαoA Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13–dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13 activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5–E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.

  • 247. Hu, Francis Jingxin
    et al.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restricted Ablation In vitro) for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improve- ments in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of intact antibody scFv genes and simultaneous diversification of all six CDRs. Here, we de- scribe the generation of mutagenesis libraries for antibody affinity maturation, using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. This procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, and elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with di- versity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed >99% functional diversity in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed quicker enrichment of improved binders compared to the other two diversification strategies.

  • 248.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab. Tech Univ Denmark, Novo Nord Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping2019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 6, artikel-id e34Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • 249.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.

  • 250.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody2014Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, nr 1, s. 35-43Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

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