Secretome profiling of apheresis platelet supernatants during routine storage via antibody-based microarray

Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

“…showing that these proteins had more than two-fold increase in their expression profiles in the "thrombinactivated" vs "resting" platelets. We recovered a high overlap of proteins members with the kinome family reported already by other proteomic analyses, [20][21][22][23][24][25][26][27][28][29][30] that are also indexed in many other research studies related to the proteomic profiling of agonists activated platelets, such as the "Adhesome" and "Platelet Web" databases, as described later in the results section. Other major pathways predicted by IPA to be up-regulated in the proteome of "thrombin-activated platelets" were mapped to the cytoskeleton, actin and RhoA mediated cellular signaling ( Figure 18).…”

“…The "in solution" digestion with three enzymes (trypsin/LysC/Glu-C), enabled the extraction of peptides from the total platelet proteome, including the membrane-derived and microvesicles contained in the releasates, as reported previously by other studies on platelets proteomics. [20][21][22][23][24][25][26][27][28][29][30] The extracted peptides were subjected to nano LC/MS/MS sequencing on a Q Exactive quadrupole orbitrap mass spectrometer, as summarized in the protocol shown in Figure 1. The label-free proteomics analysis employed the label free quantification (LFQ) module provided by PEAKS 8.5 (Bioinformatics Solutions Inc.), In addition, the changes in the protein expression profiles were complemented by an additional analysis involving the Scaffold and per SPECtives software (Proteome Software Inc.).…”

“…Research conducted in the last five years acknowledges the identification of 5000-5500 expressed proteins in human platelets, a proteome that is highly similar between different healthy individuals. [10][11][12][13][14][15][16][17][18][19][20][21] Recent emerging analytical technologies coupling the fractionation of the cellular proteome with the multidimensional nano LC/ESI/MS/MS sequencing, together with systems biology approaches empowered by bioinformatics analysis and data mining enabled a revolution in platelet proteomics that led to the development of new, mass spectrometric-based assays for enhanced research of platelet pathophysiology. [15][16][17][18][19][20][21] Consequently, the modern proteomics approaches can reveal: (1) targeted and untargeted quantitative changes in the abundances of thousands of proteins; (2) post-translational modifications; (3) protein-protein interactions networks; (4) subcellular localization, i.e.…”

“…[13][14][15][16] Using highly sensitive and highly specific mass spectrometers (providing mass accuracy and resolution), and proteome fractionations assays involving denaturant, and reducing one-dimensional or two-dimensional protein electrophoretic systems (1-DE and 2-DE, respectively) followed by protein digestions with multiple enzymes (trypsin, LysC, Glu-C and Asp-N, among others), and further fractionations of peptides by means of orthogonal chromatographic systems, thousands of proteoforms can be characterized from highly pure fractions consisting of only 2x10 8 platelets, purified from small volumes of blood, usually in the range of few milliliters. [20][21][22][23][24][25][26][27][28] Consequently, the future advances in the mass spectrometric profiling of protein expression and their corresponding highly-regulated PTM are expected to allow platelet proteomics to be implemented in many research and clinical laboratories as a reliable tool for characterizing the molecular and cellular pathways that affect platelet homeostasis in healthy and disease states. Many original research articles and recent reviews highlighted the wide area of research dedicated to label or label-free, targeted or untargeted proteomics profiling of changes in the platelets purified from patients experiencing cardiovascular, inflammatory, and bleeding disorders.…”

Platelet rich plasma in osteoarthritis: More than a growth factor therapy

“…Platelets are non-nucleated cells derived from very large pre-cursor cells called megakaryocytes that reside in the bone marrow. During their development, platelets obtain large numbers of storage granules that contain different growth factors, cytokines, and hormones required for activating acute inflammation, which is the first stage of wound repair [11,12,14,37,38] . Platelet activation is a highly regulated process that culminates in degranulation, or the release of granule contents [12,[39][40][41][42][43] .…”