Fig. 1.

Rab10-pThr73 as a readout for LRRK2 activity in human peripheral blood neutrophils.A, Workflow for global proteome analysis of human peripheral blood neutrophils. B, Proteins ranked according to their abundances across the global neutrophil proteome. Quartiles are indicated with dashed lines. LRRK2 phosphorylated Rab proteins and other PD-associated proteins are highlighted in red and blue, respectively. C, Western blot analysis of neutrophils (−/+ 200 nm MLi-2, 30 min) using anti-MJFF-pRAB10 (pThr73), Rab10 total and GAPDH antibodies. D, Heat map of z-scored phosphopeptide intensities of LRRK2-phosphorylated Rab proteins from pRab immunoprecipitation of neutrophil lysates (−/+ 200 nm MLi-2, 30 min, 3 technical replicates, n = 2). Missing values are in grey. E, Targeted MS-quantified Rab10-pThr73 and Rab43-pThr82 peptide intensities in immunoprecipitations of individual Rab10 and Rab43 proteins from neutrophils (−/+ 200 nm MLi-2, 30 min). P-values were assessed using unpaired Student's t test analysis. F, Limit of detection (LOD) of SIL Rab10-pThr73 tryptic peptide (FHpTITTSYYR) with various acquisition methods; full MS, SIM, mxSIM and PRM. Linear ranges of the dilution curves were assessed with singlets analyses of the SIL Rab10-pThr73 phosphopeptide (10 amol to 50 fmol, spiked-in a digest of HeLa). G, Limit of quantification (LOQ) for the Rab10-pThr73 tryptic peptide measured in mxSIM mode. 25 fmol of light phosphopeptide was mixed with variable amounts of its SIL counterpart (10 amol to 50 fmol) and spiked into a background of HeLa digest (3 technical replicates). Median ratios extracted from Skyline were plotted against the expected ratios.