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High-throughput protein analysis using mass spectrometry-based methods
KTH, School of Biotechnology (BIO), Protein Technology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers.

The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , x, 121 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:15
Keyword [en]
Proteomics, mass spectrometry, affinity proteomics, immunoenrichment, immunoprecipitation, IMAC, screening, protein production, protein purification, ISET, quantification, SILAC, stable isotope standard, antibody validation
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-154513ISBN: 978-91-7595-292-5 (print)OAI: oai:DiVA.org:kth-154513DiVA: diva2:757226
Public defence
2014-11-14, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20141022

Available from: 2014-10-22 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved
List of papers
1. Parallel production and verification of protein products using a novel high-throughput screening method
Open this publication in new window or tab >>Parallel production and verification of protein products using a novel high-throughput screening method
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2011 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 8, 1018-1025 p.Article in journal (Refereed) Published
Abstract [en]

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase (R) cultivation technology was applied, which enables cultivation in as small volumes as 150 mu L. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

Keyword
His-tag, IMAC, Micro-scale, Protein production, Protein purification, Screening
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-39519 (URN)10.1002/biot.201000430 (DOI)000294108100012 ()
Funder
Knut and Alice Wallenberg Foundation
Available from: 2011-09-20 Created: 2011-09-12 Last updated: 2017-12-08Bibliographically approved
2. Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
Open this publication in new window or tab >>Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
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2012 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 20, 8663-8669 p.Article in journal (Refereed) Published
Abstract [en]

A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.

Keyword
Assisted-Laser-Desorption/Ionization, Selective Enrichment Target, Spectrometry-Based Proteomics, Mass-Spectrometry, Sample Preparation, Affinity-Chromatography, Recombinant Proteins, Microfluidic Chips, Identification, Expression
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-107629 (URN)10.1021/ac3017983 (DOI)000309805200034 ()2-s2.0-84869486988 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, VR 2009-5361 VR/Vinnova/SSF MTBH 2006-7600Vinnova, 2007-02614
Note

QC 20121217

Available from: 2012-12-17 Created: 2012-12-14 Last updated: 2017-12-06Bibliographically approved
3. Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomics
Open this publication in new window or tab >>Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomics
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

An important topic of discussion in proteomics is the degree of correlation of RNA and protein levels in cells, tissues and organs. In this study, the difference in protein and mRNA levels for a number of selected gene targets were investigated across six human cell lines using quantitative proteomics and next generation sequencing-based transcriptomics. The copy numbers of 32 proteins were determined using an absolute quantitative proteomics approach (PrEST-SILAC), where heavy isotope-labeled protein fragments were used as internal standards. A cross evaluation of protein copy numbers determined by mass spectrometry and staining profiles using immunohistochemistry showed good correlation. The mRNA levels were determined using RNA sequencing based on digital counting of sequencing reads and the levels determined as FPKM values. Comparison of the relative variations in mRNA and protein levels for individual genes across the six cell lines showed correlation between protein and mRNA levels, including six genes with high variability in expression levels in the six cell lines resulting in an average correlation of 0.9 (Spearman's rank coefficient). In summary, the analysis of the selected protein targets supports the conclusion that the translation rate across cell lines correlates for a particular gene, suggesting that individual protein levels can be predicted from the respective mRNA levels by defining the relation between protein and mRNA, specific for each human gene.

National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-154507 (URN)
Note

QS 2015

Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved
4. Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry
Open this publication in new window or tab >>Investigating the Applicability of Antibodies Generated within the Human Protein Atlas as Capture Agents in Immunoenrichment Coupled to Mass Spectrometry
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 10, 4424-4435 p.Article in journal (Refereed) Published
Abstract [en]

For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2014
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-154501 (URN)10.1021/pr500691a (DOI)000342719200018 ()2-s2.0-84907810099 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation
Note

QC 20141106

Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2017-12-05Bibliographically approved
5. 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

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