Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils

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 20 ml 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.