Mass Spectrometric Phosphoproteome Analysis of Small-Sized Samples of Human Neutrophils

Clin Chim Acta. 2015 Dec 7;451(Pt B):199-207. doi: 10.1016/j.cca.2015.09.030. Epub 2015 Oct 3.

Abstract

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.

Keywords: Extracellular signal-regulated kinase 1/2; Neutrophil; Phosphoproteomics; Protease inhibition; Protein degradation; TiO(2)-MOAC enrichment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HEK293 Cells
  • Humans
  • Mass Spectrometry
  • Molecular Weight
  • Neutrophils / chemistry*
  • Phosphopeptides / blood*
  • Proteomics*

Substances

  • Phosphopeptides