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

ABSTRACT: Comprehensive characterization of platelets involves various different functional assays and analysis techniques, each requiring individual aliquots of sample. Consequently, the available sample material is often limited rendering more effective sample use highly important. Our re-cently introduced proteomics workflow for semi-automated sample preparation, involving pos-itive pressure filter-aided sample preparation (FASP) in 96-well format (PF96), allows for highly robust platelet sample preparation but displays substantial analyte loss when processing minute protein amounts in the lower µg-range. Here we describe a downscaling of PF96 to 384-well format (PF384) in terms of required starting material. We determined meaningful sample loads and highlight advantages when processing only 3 µg purified platelet protein from 22 healthy donors, namely: (I) improved identification and analyte recovery 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) Reduction of variance when de-tecting disease markers by 6 %. Hence, these advantages, in conjunction with excellent through-put capabilities, render PF384 a promising future in clinical research and might even pave the way of platelet proteomics into molecular diagnostics.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Platelet

SUBMITTER: Stefan Loroch

LAB HEAD: Albert Sickmann

PROVIDER: PXD028316 | Pride | 2025-02-14

REPOSITORIES: Pride

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			Action	DRS
	INFO_Loroch_Panagiotidis_etal.txt	Txt
	Std-QExactive01-04391_384_DW_CVD-0931.msf	Msf
	Std-QExactive01-04391_384_DW_CVD-0931.raw	Raw
	Std-QExactive01-04392_384_DW_CVD-0953.raw	Raw
	Std-QExactive01-04393_384_DW_CVD-0982.raw	Raw

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Project description:Rituximab (RTX) is widely used as a first-line therapeutic strategy in B cell-mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Primary immune thrombocytopenia (ITP) is a prototypic B cell mediated autoimmune disease in which pathogenic antibodies directed against the platelet membrane glycoprotein IIb-IIIa (GPIIbIIIa) lead to platelet destruction by macrophages in the spleen. In ITP, RTX-mediated B-cell depletion leads to an immediate clinical response in 50% of patients but 80% of patients will subsequently relapse in the next following months. Patients failing to respond or relapsing after RTX treatment are splenectomized when alternative therapies are inefficient or unavailable. Therapeutic splenectomy, resulting in a durable platelet response in 60-70 % of ITP patients, offers a unique access to study the autoimmune response in the spleen. To understand the cellular basis of disease's relapse in secondary lymphoid organs in humans, memory B cells were sorted and analyzed by scRNAseq from the spleen of three different groups of splenectomized ITP patients: 1) Patients that achieved a complete response after a course of RTX and relapsed during B-cell reconstitution (hereafter referred to as “RTX relapse” patients), 2) patients with primary failure of RTX, i.e. who did not respond to treatment at the time of B-cell depletion (“RTX failure” patients), 3) patients with active ITP that were not treated with RTX (“ITP” patients). All were compared with sorted splenic memory B cells from organ donors with no immune disease who died from stroke or head trauma, hereafter referred to as healthy donors (“HD” patients). We also included in this analysis splenic memory B cells from children with sickle cell disease that underwent splenectomy and are known to have a high frequency of B cell activation secondary to classical childhood infections and vaccinations, reflected by an increased number of GC. Single-cell mRNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016, Cell Systems 3, 385–394), with cDNA libraries generation, sequencing and reads alignment performed at Single Cell Discoveries (Utrecht, Netherlands).

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

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