Middle-throughput LC-MS-based platelet proteomics with minute sample amounts using semi-automated positive pressure FASP in 384-well format (PF384).

Comprehensive characterization of platelets requires various functional assays and analysis techniques, including omics-disciplines, each requiring an individual aliquot of a given sample. Consequently, the sample material per assay is often highly limited rendering downscaling a prerequisite for effective sample exploitation. Here we present a transfer of our recently introduced 96-well-based proteomics workflow (PF96) into the 384-well format (PF384) allowing for a significant increase in sensitivity when processing minute platelet protein amounts. In addition, the 4-fold higher throughput (1500 samples per lab worker per week) allows to easily meet the throughput capacities of modern LC-MS instruments. We determined optimal sample loads followed by highlighting the strengths in comparison to our previous sample preparation approach by processing only 3 µg of purified platelet protein from 22 healthy donors. Major advantages are: (I) improved identification and analyte recovery, especially of low copy number proteins, with signal intensity gains of +130 % and +107 % (peptide and protein level, respectively) (II) substantial intensity gains for key-players in platelet activation including the membrane receptors PAR4, P2X1, GPVI, GPV, GPIX and the downstream mediators AKT, PKA, Rap1, Lyn (III) improved reproducibility with a reduction of technical variance from 22 / 25 % down to 16 / 19 % for detection of lower / higher abundant disease markers and (IV) a 4-fold increase in sample preparation throughput. Taken together, these advantages render PF384 a promising future in clinical proteomics and might pave the way of platelet proteomics with minute sample amounts into molecular diagnostics.