Journal
CLINICA CHIMICA ACTA
Volume 451, Issue -, Pages 199-207Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.cca.2015.09.030
Keywords
Extracellular signal-regulated kinase 1/2; TiO2-MOAC enrichment; Neutrophil; Phosphoproteomics; Protein degradation; Protease inhibition
Categories
Funding
- Red Cross Blood Donation Service West, Hagen
- Germany
- Deutsche Forschungsgemeinschaft [GR 2232/7-1, GRK1870/1]
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Background: 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). Methods: Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed. Results: 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 20 ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored. Conclusion: We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies. (C) 2015 Elsevier B.V. All rights reserved.
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