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High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
2015 (Engelska)Ingår i: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, s. 68-75Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

Ort, förlag, år, upplaga, sidor
2015. Vol. 3, s. 68-75
Nyckelord [en]
Cerebrospinal fluid, Dog, High-abundant protein depletion, Shotgun proteomics, Orbitrap, Mass spectrometry
Nationell ämneskategori
Analytisk kemi
Identifikatorer
URN: urn:nbn:se:uu:diva-259611DOI: 10.1016/j.bbrep.2015.07.013OAI: oai:DiVA.org:uu-259611DiVA, id: diva2:844987
Tillgänglig från: 2015-08-10 Skapad: 2015-08-10 Senast uppdaterad: 2015-10-01Bibliografiskt granskad
Ingår i avhandling
1. Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Öppna denna publikation i ny flik eller fönster >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2015. s. 82
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Nyckelord
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
Nationell ämneskategori
Analytisk kemi
Forskningsämne
Kemi med inriktning mot analytisk kemi
Identifikatorer
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Disputation
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Svenska)
Opponent
Handledare
Tillgänglig från: 2015-09-03 Skapad: 2015-08-10 Senast uppdaterad: 2015-10-01

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Sundberg, MårtenBergquist, JonasRamström, Margareta

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