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Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
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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.

Place, publisher, year, edition, pages
2012. Vol. 84, no 20, 8663-8669 p.
Keyword [en]
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
URN: urn:nbn:se:kth:diva-107629DOI: 10.1021/ac3017983ISI: 000309805200034ScopusID: 2-s2.0-84869486988OAI: diva2:577775
Knut and Alice Wallenberg FoundationSwedish Research Council, VR 2009-5361 VR/Vinnova/SSF MTBH 2006-7600Vinnova, 2007-02614

QC 20121217

Available from: 2012-12-17 Created: 2012-12-14 Last updated: 2014-10-22Bibliographically approved
In thesis
1. High-throughput protein analysis using mass spectrometry-based methods
Open this publication in new window or tab >>High-throughput protein analysis using mass spectrometry-based methods
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.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:15
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
urn:nbn:se:kth:diva-154513 (URN)978-91-7595-292-5 (ISBN)
Public defence
2014-11-14, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)

QC 20141022

Available from: 2014-10-22 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved

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