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Automated sample preparation method for mass spectrometry analysis on recombinant proteins
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0003-0605-8417
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2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 20, 4457-4464 p.Article in journal (Refereed) Published
Abstract [en]

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5 h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000-35,000 Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization anti matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

Place, publisher, year, edition, pages
2009. Vol. 1216, no 20, 4457-4464 p.
Keyword [en]
Mass spectrometry, High-throughput, Automated sample preparation, Recombinant proteins, IMAC, RPLC, affinity-chromatography, purification
URN: urn:nbn:se:kth:diva-18416DOI: 10.1016/j.chroma.2009.03.041ISI: 000265956100021OAI: diva2:336463
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-01-21Bibliographically approved
In thesis
1. Characterization of Antigenic Properties and High Throughput Protein Purification
Open this publication in new window or tab >>Characterization of Antigenic Properties and High Throughput Protein Purification
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.


In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.


The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors.


Place, publisher, year, edition, pages
Stockholm: KTH, 2010. 90 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2010:19
high throughput, protein purification, sample preparation, antigen solubility, immunogenicity, epitope mapping, antigen design
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-25841 (URN)978-91-7415-768-0 (ISBN)
Public defence
2010-11-12, The Svedbergsalen, Roslagstullsbacken 21, AlvaNova, Stockholm, 10:00 (Swedish)
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-02Bibliographically approved

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Steen, JohannaRamström, MargaretaUhlén, MathiasHober, SophiaOttosson, Jenny
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