Journal article
Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils
Clinica chimica acta, Vol.451(Pt B), pp.199-207
12/07/2015
DOI: 10.1016/j.cca.2015.09.030
PMID: 26434552
Abstract
Global analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC).
Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed.
Degradation of proteins in neutrophil extracts was observed after preparation with a urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl–methionyl–leucyl–phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored.
We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.
•Neutrophil protein extracts are unstable in buffers containing 8mol/l urea.•Highly concentrated protease inhibitors stabilize neutrophil extracts in urea.•The modified lysis buffer does not interfere with tryptic digestion.•PolyMAC-Ti sensitively enriches neutrophil phosphopeptides from 200μg of sample.•The workflow enables gel-free phosphoproteomics of neutrophils with 20ml of blood.
Details
- Title: Subtitle
- Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils
- Creators
- Stefan Muschter - Universitätsmedizin GreifswaldTom Berthold - Universitätsmedizin GreifswaldGourav Bhardwaj - Universitätsmedizin GreifswaldElke Hammer - Universitätsmedizin GreifswaldVishnu Mukund Dhople - Universitätsmedizin GreifswaldJan Wesche - Universitätsmedizin GreifswaldAngelika Reil - German Red CrossJürgen Bux - Ruhr University BochumTamam Bakchoul - Universitätsmedizin GreifswaldLeif Steil - Universitätsmedizin GreifswaldAndreas Greinacher - Universitätsmedizin GreifswaldUwe Völker - Universitätsmedizin Greifswald
- Resource Type
- Journal article
- Publication Details
- Clinica chimica acta, Vol.451(Pt B), pp.199-207
- Publisher
- Elsevier B.V
- DOI
- 10.1016/j.cca.2015.09.030
- PMID
- 26434552
- ISSN
- 0009-8981
- eISSN
- 1873-3492
- Grant note
- name: Red Cross Blood Donation Service West, Hagen; Germany; DOI: 10.13039/501100001659, name: Deutsche Forschungsgemeinschaft, award: GR 2232/7-1, GRK1870/1
- Language
- English
- Date published
- 12/07/2015
- Academic Unit
- Internal Medicine
- Record Identifier
- 9984359827802771
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