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Targeted proteomics methods for protein quantification of human cells, tissues and blood
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Uhlén lab)ORCID iD: 0000-0002-0017-7987
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.

                     

The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.

                     

In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator.

Place, publisher, year, edition, pages
Stockholm: Kungliga Tekniska högskolan, 2016. , 90 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:16
Keyword [en]
proteomics, mass spectrometry, protein quantification, stable isotope standard, parallel reaction monitoring, immuno-enrichment
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-193951ISBN: 978-91-7729-153-4 (print)OAI: oai:DiVA.org:kth-193951DiVA: diva2:1034873
Public defence
2016-11-11, Gard-aulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-14Bibliographically approved
List of papers
1. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins
Open this publication in new window or tab >>Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins
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2014 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, 1611-1624 p.Article in journal (Refereed) Published
Abstract [en]

AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-147956 (URN)10.1074/mcp.M113.034140 (DOI)000337239500018 ()2-s2.0-84901952830 (Scopus ID)
Note

QC 20150217

Available from: 2014-07-11 Created: 2014-07-10 Last updated: 2017-12-05Bibliographically approved
2. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
Open this publication in new window or tab >>Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
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2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, e49Article in journal (Refereed) Published
Abstract [en]

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

Keyword
PCR Products, Restriction Enzymes, Magnetic Beads, In-Vitro, One-Pot, Protein, Polymerase, Expression, Construction, Proteomics
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-159278 (URN)10.1093/nar/gkv036 (DOI)000354722500007 ()25618848 (PubMedID)2-s2.0-84961523206 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceNovo NordiskKnut and Alice Wallenberg FoundationVINNOVA
Note

QC 20150203

Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
3. Gene specific correlation of RNA and protein levels in human cells and tissues
Open this publication in new window or tab >>Gene specific correlation of RNA and protein levels in human cells and tissues
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2016 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292Article in journal (Refereed) In press
Abstract [en]

An important issue for molecular biology is to establish if transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on Parallel Reaction Monitoring to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP) conversion factor independent of the tissue-type is introduced, thus significantly enhancing the predictability of protein copy numbers from RNA levels. The results show that the RTP-ratio varies significantly with a few hundred copies per mRNA molecule for some genes to several hundred thousands protein copies per mRNA molecule for others. In conclusion, our data suggests that transcriptome analysis can be used as a tool to predict the protein copy numbers per cell, thus forming an attractive link between the field of genomics and proteomics. 

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193966 (URN)
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2017-11-29Bibliographically approved
4. Validation of antibodies for Western blot applications using orthogonal methods
Open this publication in new window or tab >>Validation of antibodies for Western blot applications using orthogonal methods
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

There is a great need for standardized validation methods for antibody specificity and selectivity. Here, we describe the use of orthogonal methods in which the specificity of an antibody in a particular application is determined based on correlation of protein abundance across several samples using an antibody-independent method. We show that pair-wise correlation between orthogonal samples can be used to score the specificity of antibodies in a standardized manner using a test panel of human cell lines. Here, we investigated two independent methods for validation of antibodies in Western blot applications, namely transcriptomics and targeted proteomics and we show that the two methods yield similar, but not identical results. The orthogonal methods can also be used to investigate on- and off- target binding for antibodies with multiple bands in the Western blot assay. In conclusion, orthogonal methods for antibody validation provide an attractive strategy for systematic validation of antibodies in a quantitative manner. 

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193962 (URN)
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-13Bibliographically approved
5. A recombinant protein standard resource for targeted proteomics
Open this publication in new window or tab >>A recombinant protein standard resource for targeted proteomics
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here, we have used a resource of 26,000 recombinant protein fragments to create custom libraries of standards for targeted proteomics based on parallel reaction monitoring (PRM). The recombinant fragments can be produced in a bacterial cell factory to generate heavy isotope labeled standards for absolute quantification of the corresponding protein targets and be used to produce high- quality spectral libraries. Altogether, coordinates for 25,684 unique proteotypic peptide assays have been experimentally defined covering 10,163 human proteins. The protocol allows for precise monitoring of digestion kinetics and thus enables to select peptides that behave quantitative during the sample preparation process. We show that the quantification tag of each recombinant protein fragment can be used for accurate retention time prediction and allows for assay standardization across different method parameters. The use of this resource was illustrated by determining the absolute concentrations of selected protein targets using multiplex targeted proteomics assays for determination of quantitative assessment of 49 protein targets in serum samples. 

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-193964 (URN)
Note

QC 20161013

Available from: 2016-10-13 Created: 2016-10-13 Last updated: 2016-10-13Bibliographically approved

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