Table 1 Proteome studies of circulating microparticles, and of in vitro generated platelet-and granulocyte-derived microparticles. Study design, methodology and main results
Study | References | Objectives | Study populationa | Pre-analytical protocol | Details | Proteome analysis workflow | Details | Main findings |
|---|---|---|---|---|---|---|---|---|
Platelet-derived MPs | Garcia et al. [49] | Characterize and compare the proteome of in vitro generated platelet-derived MPs (PMPs) to platelets | Individuals: 1 healthy donor | Sample collection | Whole-blood Anticoagulant: acid-citrate-dextrose (ACD) | Workflow | Gel-LC–MSMS shotgun workflow: 1-D gel electrophoresis (Coomassie Blue staining) with lanes cut into 26 bands and digested before LC–MS/MS | 578 proteins in total were identified in PMPs. 380 of these proteins had not previously been identified in platelets (at the time of publication) |
Sex: not stated | PRP generation (cell removal) | Cent. 110 g/15 min/T? | Tryptic digestion | In-gel digestion | ||||
Age: not stated | Plt isolation | Cent. 710 g/15 min/T? Followed by 3× wash (Cent. 2000 g/10 min/T?) | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer (with 1.5 mM CaCl2, 0.4 mM MgCl2); debris and cell removal from plt prep using an additional cent. 110 g/10 min/T? | Instrument | Finnigan LTQ Ion Trap (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | ADP | Software for protein ID | SEQUEST | |||||
Removal of activated Plts and cells | Cent. 710 g/15 min/T? Followed by 3× wash (Cent. 2000 g/10 min/T?) | Protein database | NCBInr (human) Taxonomy: human # Entries: not stated | |||||
PMP isolation | Cent. 150,000 g/90 min/10 °C (Pellet = MPs) | |||||||
Platelet-derived MPs | Piersma et al. [51] | Characterize the releasate (including MPs) from in vitro activated platelets | Individuals: 3 healthy donors | Sample collection | Whole-blood Anticoagulant: acid-citrate-dextrose (ACD) Prostaglandin E1 was added to inhibit platelet activation | Workflow | Gel-LC–MSMS shotgun workflow: 1-D gel electrophoresis (Coomassie staining) with lanes cut into 15 bands (Exp #1) or into 10 bands (Exp #2) and digested before LC–MS/MS | 716 proteins in total were identified in the platelet releasate 225 proteins were identified in the releasate from all 3 individuals and defined as the “core” releasate proteome 55% of the “core” proteome overlapped with previous publications of PMP and α-granule proteomes 45% of the “core” proteome was unique to the platelet realesate The unstimulated control showed few bands on SDS-PAGE gel and was not analysed |
Sex: not stated | PRP generation (cell removal) | Cent. 150 g/15 min/RT | Tryptic digestion | In-gel digestion | ||||
Age: not stated | Plt isolation | Cent. 720 g/10 min/RT Followed by 2× wash in Tyrode’s buffer | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer | Instrument | LTQ-FT hybrid mass spectrometer (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | Thrombin receptor activating peptide, non-stimulated platelets as controls | Software for protein ID | SEQUEST | |||||
Removal of activated Plts and cells | 2× Cent. 1000 g/10 min | Protein database | IPI.human v3.31 Taxonomy: human # Entries: 67,511 | |||||
Plt releasate isolation | Supernatant from plt removal was concentrated on Amicon Ultra-4 cell (10 kDa cut-off) | |||||||
Platelet-derived MPs | Dean et al. [52] | Characterize the proteome of in vitro generated platelet-derived MPs of different sizes | Individuals: 7–10 healthy donors | Sample collection | Whole-blood Anticoagulant: citrate Samples were pooled before plt isolation | Workflow | Shot-gun proteomics workflow | Total number of identified proteins based on size-exclusion fractions: 54 (#1), 49 (#2), 293 (#3), and 150 (#4) Proteins were highly differentially expressed between the four fractions Mitochondrial proteins were enriched in fraction 1 Cytoskeletal proteins were enriched in fractions 3 and 4 and more similar to plasma membranes |
Sex: not stated | PRP generation (cell removal) | 2× Cent. 150 g/10 min/RT | Tryptic digestion | In-solution digestion | ||||
Age: not stated | Plt isolation | 2× Cent. 1500 g/10 min/RT | Method | 2D-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer (calcium free) | Instrument | LTQ Plus Ion Trap (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | CaCl2, thrombin, collagen | Software for protein ID | SEQUEST | |||||
Removal of activated Plts and cells | Cent. 5000 g/t?/T? | Protein database | Human Ref-Seq Taxonomy: Human # Entries: not stated | |||||
PMP isolation | Cent. 130,000 g/t?/T? (Pellet = MPs) Pellet were then subject to gel-filtration to obtain 4 different PMP size fractions | |||||||
Platelet-derived MPs | Shai et al. [46] | Characterize in vitro generated platelet-derived MP proteomes according to different platelet activation stimuli | Individuals: 4 healthy donors | Sample collection | Fresh platelets were collected using an apheresis system (MCS platelet collection system) and washed (solution containing citric acid, PGE1 and apyrase) | Workflow | 2-D gel electrophoresis (Sypro Ruby staining), differential image analysis to identify differentially expressed proteins and ID of spots of interest using MS | 26 proteins were identified as differentially expressed between thrombin induced and shear-stress induced MPs |
Sex: not stated | PRP generation (cell removal) | See sample collection | Tryptic digestion | In-gel digestion | ||||
Age: 20–35 yrs | Plt isolation | See sample collection | Method | MALDI-TOF/TOF or LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Buffer not stated | Instrument | 4800 MALDI-TOF/TOF (Applied Biosystems) Amazon ETD Ion Trap (Bruker) | ||||
Plt activation for PMP in vitro generation | Thrombin or shear stress | Software for protein ID | Mascot v2.1 and Mascot v2.3 | |||||
Removal of activated Plts and cells | 2× Cent. 1500 g/t?/T? | Protein database | SwissProt release 56.0 Taxonomy: Human Entries: not stated SwissProt release 57.15 Taxonomy: Human # Entries: not stated | |||||
PMP isolation | Cent. 100,000 g/60 min/T? (Pellet = MPs) | |||||||
Platelet-derived MPs | Capriotti et al. [56] | Comparative analysis of the proteome of in vitro generated PMPs using a shot-gun proteomic protocol to analyze in-solution digested proteins obtained with or without an additional hydrogel nanoparticle (HN) enrichment protocol for low molecular weight proteins | Individuals: 12 healthy donors | Sample collection | Whole blood. Anti-coagulant: K2EDTA and protease inhibitors cocktail. Samples were pooled before Plt isolation | Workflow | Shot-gun proteomics workflow | In total 603 proteins were identified combining identifications obtained with or without the HN enrichment step 318 proteins were exclusively identified using the standard procedure and 57 proteins were only identified by including the HN enrichment step, while the remaining 228 proteins were identified using both methods A higher proportion of low molecular weight proteins were found in the HN protocol Compared to the PMP proteome in Garcia et al. (2005), 360 proteins overlapped, while 243 did not overlap. Of the 243 proteins, 130 had previously been confined to the platelet proteome |
Sex: All males | PRP generation (cell removal) | Cent. 110 g/15 min/T? | Tryptic digestion | Proteins precipitated using chloroform/methanol method before in-solution digestion | ||||
Age: 20–40 yrs | Plt isolation | Cent. 710 g/15 min/T? Followed by 3× wash (Cent. 2000 g/10 min) | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer (with 1.5 mM CaCl2, 0.4 mM MgCl2); debris and cell removal from plt prep using an additional cent. 110 g 10 min | Instrument | LTQ-Orbitrap XL (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | ADP | Software for protein ID | Proteome Discoverer v1.2 and Mascot v 2.3.2 Protein IDs validated in Scaffold v3.1.2 | |||||
Removal of activated Plts and cells | Cent. 710 g/15 min/T? | Protein database | SwissProt release 57.15 Taxonomy: Human # Entries: 20,266 | |||||
PMP isolation | Cent. 150,000 g/90 min/4 °C (Pellet = MPs) | |||||||
Platelet-derived MPs | Milioli et al. [41] | Quantative proteome analysis of in vitro generated platelet MPs formed by platelet activation using agonists of increasing potency | Individuals: 3 healthy donors | Sample collection | Fresh platelets were collected using an apheresis system | Workflow | Shot-gun proteomics workflow, Differentially expressed proteins identified using the iTRAQ method | 3383 proteins were identified 1109 proteins were identified in all three biological replicates Significant differential expression of proteins dependent on the stimuli was observed |
Sex: not stated | PRP generation (cell removal) | See above | Tryptic digestion | In-solution digestion | ||||
Age: not stated | Plt isolation | Cent. 700 g/20 min/20 °C Followed by 2× wash in Tyrode’s buffer (with 1 mM EDTA) | Method | iTRAQ labelleing of peptides was followed by HILIC fractionation before analysis by nano-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer (pH 6) | Instrument | Q-Exactive Plus (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | (a) 10 uM ADP (b) 1 U/mL thrombin (c) 20 ug/mL collagen (d) 1 U/mL thrombin and 20 mg/mL collagen | Software for protein ID | Proteome Discoverer v1.4.0.288 in combination with Mascot v2.3 and SEQUEST HT | |||||
Removal of activated Plts and cells | Cent. 710 g/20 min/20 °C Cent. 1000 g/20 min/20 °C | Protein database | SwissProt release v3.53 Taxonomy: Human # Entries: 20,243 | |||||
PMP isolation | Cent. 130,000 g/60 min/4 °C (Pellet = MPs) | |||||||
Plasma MPs | Little et al. [39] | Characterize the plasma MP proteome in a general population | Individuals: 42 patients diagnosed with: (a) Cardiovascular disease (b) Hypertension (c) Diabetes mellitus (d) Cancer | Sample collection | Whole-blood Anti-coagulant: acid-citrate-dextrose | Workflow | Shot-gun proteomics workflow | 458 proteins were identified 130 proteins were identified in 50% of the samples and defined as the “core” proteome Both “core” and non-”core” protein expression were correlated to age and gender. Correlates to comorbidities were not stated |
Age: 61–77 yrs (avg 69.5 yrs) | PRP generation (cell removal) | Cent. Unknown g/15 min/RT | Tryptic digestion | In-solution digestion | ||||
Sex: 12 female, 30 males | PPP/PFP generation (Plt removal) | 2× Cent. 2000 rpm (Unknown g)/15 min/RT | Method | Nano-LC–MS/MS | ||||
Ethnicity: 40 Caucasian, 2 African-American | Storage of PPP/PFP before MP isolation | Immediate processing of PPP to isolate MPs | Instrument | LTQ-FT hybrid mass spectrometer (Thermo Fisher Scientific) | ||||
Other: 7 Smokers | MP isolation | Gel filtration (not further specified) followed by cent. 100,000 g/120 min/T? | Software for protein ID | SEQUEST | ||||
Protein database | IPI.human (version not stated) Taxonomy: human # Entries: not stated | |||||||
Plasma MPs | Ramacciotti et al. [12] | Characterize the plasma MP proteome in patients with deep venous thrombosis (DVT) compared to controls | Individuals: (a) 9 patients with DVT (b) 9 patients with leg pain without DVT (c) 6 healthy controls | Sample collection | Whole-blood Anti-coagulant: acid-citrate-dextrose | Workflow | Shot-gun proteomics workflow, Differentially expressed proteins identified using the iTRAQ method | 151 proteins were identified 35 proteins displayed enrichment or depletion in plasma MPs in DVT patients in two out three experiments Alpha-2-macroglobulin and galectin-3-binding protein (G3BP) were the only enriched proteins in all DVT patients 11 proteins were depleted in all three experiments in plasma MPs in DVT patients |
Sex: not stated | PRP generation (cell removal) | NA (PPP was generated directly from whole blood) | Tryptic digestion | In-solution digestion | ||||
Age: not stated | PPP/PFP generation (Plt removal) | Cent. 1500 g/25 min/RT followed by 2× Cent. 15,000 g/2 min/RT resulting in PFP | Method | iTRAQ labelleing of peptides was followed by SCX fractionation before analysis by nano-LC–MS/MS | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | PFP was stored at −70 °C (12–24 mo) before MP isolation | Instrument | 4800 MALDI-TOF/TOF (Applied Biosystems) | ||||
MP isolation | Cent. 200,000 g/120 min/4 °C Followed by 1× wash in 0.25 M KBr and Cent. 200,000 g/120 min/4 °C | Software for protein ID | GPS Explorer v3.6 in combination with Mascot v2.1 | |||||
Protein database | NCBInr Taxonomy: mammals # Entries: not stated | |||||||
Plasma MPs | Ostergaard et al. [40] | Characterize the plasma MP proteome. Evaluate the MP washing procedure, reproducibility, and compare analysis of pooled to individual samples | Individuals: 12 healthy donors | Sample collection | Whole blood Anti-coagulant: sodium citrate | Workflow | Shot-gun proteomics workflow, differentially expressed proteins identified by label-free quantitation | 536 proteins were identified High analytical reproducibilty Significant overlap between pools and single samples 334 proteins were identified in 50% of the samples and were defined as the healthy “core” proteome Four washing steps were needed to purifiy plasma MPs |
Sex: 8 female, 4 male | PRP generation (cell removal) | Cent. 1800 g/10 min/21 °C | Tryptic digestion | In-solution digestion | ||||
Age: 24–62 yrs (avg 41.1 yrs) | PPP/PFP generation (Plt removal) | Cent. 3000 g/10 min/21 °C | Method | Nano-LC–MS/MS | ||||
Ethnicity: All Caucasian | Storage of PPP/PFP before MP isolation | PPP was stored at −80 °C before MP isolation | Instrument | LTQ-Orbitrap XL (Thermo Fisher Scientific) | ||||
MP isolation | Cent. 18,890 g/30 min/21 °C followed by 4× wash in PBS-citrate (154 mM NaCl, 1.4 mM phosphate, 10.5 mM trisodium citrate, pH 7.4) and 4X Cent. 18,890 g/30 min/21 °C (Pellet = MPs) | Software for protein ID | MaxQuant v1.1.1.25 with the built-in Andromeda search engine | |||||
Protein database | IPI.human.v3.68.fasta Taxonomy: human # Entries: 87,061 | |||||||
Plasma MPs | Bastos-Amador et al. [48] | Characterize the plasma MP proteome from healthy controls | Individuals: 38 healthy donors | Sample collection | Not stated (Plasma was obtained from a blood bank) | Workflow | Shot-gun proteomics workflow | 161 microparticle-associated proteins were identified 52 of the identified proteins belong to the immunoglobulin family Large variability was observed in the protein complement identified from MPs isolated from different donors |
Sex: 11 males, 27 females | PRP generation (cell removal) | Not stated | Tryptic digestion | In-solution digestion | ||||
Age: 20–58 yrs (avg 40.9 yrs) | PPP/PFP generation (Plt removal) | Cent. 2000 g/30 min/T? followed by Cent. 12,000 g/45 min/T? | Method | Nano-LC–MSE | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | Immediate processing of PFP to isolate MPs | Instrument | Q-Tof Premier (Waters Corporation) | ||||
MP isolation | Cent. 110,000 g/120 min/T? followed by Filtering 0.22 um followed by Cent. 100,000 g/60 min/T? (Pellet = MPs) | Software for protein ID | ProteinLynx GlobalServer v 2.6 | |||||
Protein database | SwissProt (version not stated) Taxonomy: not stated # Entries: not stated | |||||||
Plasma MPs | Chaichompoo et al. [54] | Characterize plasma MPs in patients with β-thalassemia/hemoglobin E and healthy controls | Individuals: (a) 15 β-thalassemia/hemoglobin E patients (β-thal/HbE) (b) 15 healthy donors | Sample collection | Whole blood Anti-coagulant: K3EDTA, heparin, citrate | Workflow | 2-D gel electrophoresis with silver staining Differentially expressed proteins selected by image analysis (Image Master Platinum) followed by identification by mass spectrometry analysis | 1000–1200 proteins spots were identified in each 2-D gel 29 protein spots differed significantly 9 proteins were decreased in β-thal/HbE patients 12 proteins were found increased in β-thal/HbE patients 8 proteins were only detected in MPs β-thal/HbE patients |
Sex: not stated | PRP generation (cell removal) | Cent. 1500 g/15 min/20 °C | Tryptic digestion | In-gel digestion | ||||
Age: 30.9 ± 8.9 yrs (patients) Age: 25.3 ± 1.8 yrs (healthy donors) | PPP/PFP generation (Plt removal) | 2× Cent. 14,000 g/2 min/20 °C resulting in PFP | Method | MALDI Q-TOF | ||||
Ethnicity: All asians | Storage of PPP/PFP before MP isolation | Immediate processing of PFP to isolate MPs | Instrument | MALDI Q-TOF Ultima (Micromass) | ||||
MP isolation | Cent. 14,000 g/45 min/20 °C followed by 2× wash in PBS (with 0.32% citrate) and 2× Cent. 14,000 g/10 min/20 °C (Pellet = MPs) | Software for protein ID | Mascot | |||||
Protein database | NCBInr Taxonomy: not stated (All?) # Entries: 17,172,511 | |||||||
Plasma MPs | Ostergaard et al. [11] | Characterize the plasma MP proteome in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc) and healthy controls (NOR) | Individuals: (a) 12 SLE patients (b) 6 RA pateints (c) 6 SSc patients (d) 12 NOR | Sample collection | Whole blood Anti-coagulant: sodium citrate | Workflow | Shot-gun proteomics workflow, differentially expressed proteins identified by label-free quantitation | 534 proteins were identified in total 314 of the proteins were idenitified in 2/3 of the samples 191 proteins were twofold upregulated in SLE, in particular: G3BP, ficolin-2, 14-3-3, β-2-glycoprotein-I, and CD5 antigen-like protein, Immunglobulins and complement proteins 57 proteins were twofold decreased in SLE |
Sex (a) SLE: 12 females (b) RA: 2 males, 4 females (c) SSc: 6 females (d) NOR: 4 males, 8 females | PRP generation (cell removal) | Cent. 1800 g/10 min/21 °C | Tryptic digestion | In-solution digestion | ||||
Age (a) SLE: 23–52 yrs (median 34 yrs) (b) RA: 35–56 yrs (median 39 yrs) (c) SSc: 33–68 yrs (median 45 yrs) (d) NOR: 24–6 yrs (median 35 yrs) | PPP/PFP generation (Plt removal) | Cent. 3000 g/10 min/21 °C | Method | Nano-LC–MS/MS | ||||
Ethnicity: All Caucasian | Storage of PPP/PFP before MP isolation | PPP was stored at −80 °C before MP isolation | Instrument | LTQ-Orbitrap XL (Thermo Fisher Scientific) | ||||
MP isolation | Cent. 18,890 g/30 min/21 °C followed by 4× wash in PBS-citrate (154 mM NaCl, 1.4 mM phosphate, 10.5 mM trisodium citrate, pH 7.4) and 4X Cent. 18,890 g/30 min/21 °C (Pellet = MPs) | Software for protein ID | MaxQuant v1.1.1.36 with the built-in Andromeda search engine | |||||
Protein database | IPI.human.v3.68.fasta Taxonomy: Human # Entries: 87,061 | |||||||
Plasma MPs | Datta et al. [55] | Compare the plasma MP proteome in subgroups of patients with lacunar infarction (LACI) and controls | Individuals: (a) 45 LACI patients: Group I: no adverse events, n = 19 Group II: recurrent vascular events, n = 11 Group III: cognitive decline, no recurrent vascular events, n = 15 (b) 17 healthy controls | Sample collection | Whole blood Anti-coagulant: EDTA | Workflow | Gel-LC–MSMS shotgun workflow: 1-D gel electrophoresis (Stain not stated) with lanes cut into bands (Number not stated) and digested before LC–MS/MS. Differentially expressed proteins identified using the iTRAQ method | 183 proteins were identified 43 specific proteins were overexpressed compared to the healthy controls: Group I: 17, Group II: 33, Group II: 28 Of the 43 proteins myelin basic protein were most upregulated Cluster analysis revealed two groups of proteins associated with clinical outcome G3BP was decreased and together with A2M were not associated with any of the disease groups |
Sex (a) LACI patients Group I: 17 males, 2 females Group II: 8 males, 3 females Group III: 5 males, 10 females (b) Controls: 4 males, 22 females | PRP generation (cell removal) | Conditions for PRP generation not stated PRP was frozen before further work | Tryptic digestion | In-gel digestion | ||||
Age (Mean (SD)) (a) LACI patients Group I: 61 yrs (9 yrs) Group II:65 yrs (10 yrs) Group III: 66 yrs (9 yrs) (b) Controls: 56 yrs (9 yrs) | PPP/PFP generation (Plt removal) | Samples were pooled before platelet removal 2× Cent. 4000 g/30 min/T? followed by Cent 12,000 g/30 min/T? | Method | iTRAQ labelleing of peptides was followed by ERLIC fractionation before analysis by nano-LC–MS/MS | ||||
Ethnicity (a) LACI patients Group I: 17 of 19 Chinese Group II: 8 of 11 Chinese Group III: 15 of 15 Chinese (b) Controls: 17 of 17 Chinese | Storage of PPP/PFP before MP isolation | Immediate processing of PFP to isolate MPs | Instrument | QSTAR Elite Hybrid MS (Applied Biosystems) | ||||
MP isolation | Cent. 30,000 g/120 min/T? followed by Cent. 200,000 g/135 min/T? (Pellet = MPs) followed by 2× wash with PBS; Cent. not stated | Software for protein ID | ProteinPilot v 3.0 | |||||
Protein database | UniProt - version not stated Taxonomy: Human # Entries 191,242 | |||||||
Plasma and platelet-derived MPs | Jin et al. [37] | Compare the plasma MP proteome to plasma and platelet proteomes | Individuals: 16 healthy donors | Sample collection | Whole blood Anti-coagulant: sodium citrate | Workflow | 2-D gel electrophoresis (Sypro Ruby staining), differential image analysis (ImageMaster 2D) to identify differentially expressed proteins and ID of spots of interest using MS | 1021–1055 protein spots were identified in the plasma MPs 331–370 protein spots were identified in plasma (PPP) 169 overexpressed protein spots in plasma MPs were excised for protein identification 83 proteins including their isoforms were identified in plasma MPs 30 of these proteins had not been previously identified in the plasma proteome |
Sex: 8 males, 8 females | PRP generation (cell removal) | Not stated (Plasma samples were obtained frozen) | Tryptic digestion | In-gel digestion | ||||
Age: 18–65 yrs | PPP/PFP generation (Plt removal) | Cent. 3200 g/30 min/T? | Method | MALDI-TOF/TOF | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | Immediate processing of PFP to isolate MPs | Instrument | 4700 Proteomics Analyzer (Applied Biosystems) | ||||
MP isolation | Cent. 250,000 g/60 min/T? (Pellet = MPs) followed by 2× wash with PBS (Cent. not stated) | Software for protein ID | Mascot (version not stated) | |||||
Protein database | Not stated (SwissProt -derived from ID table) Taxonomy: not stated # Entries: not stated | |||||||
Plasma and platelet-derived MPs | Smalley et al. [38] | Compare the plasma MP proteome to in vitro generated platelet-derived MPs | Individuals: 3 healthy donors | Sample collection | Whole-blood Anticoagulant: acid-citrate-dextrose (ACD) | Workflow | Gel-LC–MSMS shotgun workflow 1-D gel electrophoresis (No staining) with lanes cut into 1 bands and digested before LC–MS/MS Differentially expressed proteins identified by spectral counting or using the ICAT labelling method | 19 proteins were detected in plasma MPs and not in PMPs 2 proteins (von Willebrand factor and albumin) were detected in both populations but enriched in plasma MPs 11 proteins were enriched in platelet-derived MPs |
Sex: not stated | PRP generation (cell removal) | Cent. 110 g/15 min/T? | Tryptic digestion | In-gel digestion | ||||
Age: not stated | Plt isolation | Cent. 710 g/15 min/T? (Supernatant = PPP) Followed by 3× wash (Cent. 2000 g/10 min/T?) | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Plt resuspension before activation | Tyrode’s buffer (with 1.5 mM CaCl2, 0.4 mM MgCl2); debris and cell removal from plt prep using an additional cent. 110 g/10 min/T? | Instrument | Finnigan LTQ Ion Trap (Thermo Fisher Scientific) | ||||
Plt activation for PMP in vitro generation | ADP | Software for protein ID | SEQUEST | |||||
Removal of activated Plts and cells | Cent. 710 g/15 min/T? Followed by 3× wash (Cent. 2000 g/10 min/T?) | Protein database | NCBInr (Human) Taxonomy: Human # Entries: not stated | |||||
PMP isolation | Cent. 150,000 g/90 min/10 °C (Pellet = MPs) | |||||||
Sample collection | Whole-blood. Anticoagulant: acid-citrate-dextrose (ACD) | |||||||
PRP generation (cell removal) | Cent. 110 g/15 min/T? | |||||||
PPP/PFP generation (Plt removal) | Cent. 710 g/15 min/T? (Sup. = PPP) followed by 2× Cent. 710 g/15 min/25 °C | |||||||
Storage of PPP/PFP before MP isolation | Immediate processing of PPP to isolate MPs | |||||||
MP isolation | Gelfiltration of PPP followed by cent. 150,000 g/90 min/10 °C (Pellet = MPs) | |||||||
Erythrocyte-derived MPs | Rubin et al. [50] | Characterize the proteome of MPs derived from erythrocyte concentrates (ECs) to explore the effect of blood storage | Individuals: number of donors not stated | Sample collection | Whole-blood (collected in 500 ml in blood bags) Anticoagulant: citrate–phosphate-dextrose Leukocytes and platelets were removed by filtration Plasma was removed from erythrocytes by centrifugation (conditions not stated) Erythrocytes were resuspended in sodium-adenine-glucose-mannitol solution and stored as erythrocyte concentrates (ECs) at 4 °C until further analysis | Workflow | 1-D gel electrophoresis (Coomassie Blue staining); bands of interest were out and in-gel digested MS analysis | 24 proteins were identified from selected bands from an SDS-PAGE gel lane loaded with erythrocyte membranes and 16 proteins were identified from a lane loaded with erythrocyte derived MPs Carbonic anhydrases, peroxiredoxins, and 14-3-3 proteins were abundant but not differentially expressed between membranes and MPs Stomatin, Band 3 and Rhesus protein were enriched in erythrocyte MPs |
Sex: not stated | PRP generation (cell removal) | Not applicable | Tryptic digestion | In-gel digestion | ||||
Age: not stated | PPP/PFP generation (Plt removal) | Not applicable | Method | MALDI-TOF/TOF or LC–MS/MS | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | Not applicable | Instrument | 4700 MALDI-TOF/TOF (Applied Biosystems) | ||||
MP isolation | 2× Cent. 1850 g/20 min/4 °C (of ECs) followed by Cent. 3200 g/20 min/4 °C followed by 3× Cent. 120,000 g/90 min/4 °C (Pellet = MPs) | Software for protein ID | Mascot v2.0 | |||||
Protein database | UniProt - version not stated Taxonomy: not stated # Entries: not stated | |||||||
Erythrocyte-derived MPs | Bosman et al. [53] | Compare the proteome of erythrocyte-derived plasma MPs to erythrocyte membranes | Individuals: number of donors not stated | Sample collection | Whole-blood Anticoagulant: citrate | Workflow | Gel-LC–MSMS shotgun workflow: 1-D gel electrophoresis (Coomassie Blue staining) with lanes cut into 6 bands and digested before LC–MS/MS. Differentially expressed proteins were identified using the emPAI method | 271 proteins were identified in erythrocyte-derived MPs and erythrocyte membrane fractions 71 different proteins were identified in erythrocyte-derived MPs Comparison of the differential distribution of proteins based on their subcellular localization/function |
Sex: not stated | PRP generation (cell removal) | Cent. 1550 g/7 min/20 °C | Tryptic digestion | In-gel digestion | ||||
Age: not stated | PPP/PFP generation (Plt removal) | Cent. 1550 g/7 min/20 °C | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | Immediate processing of PPP to isolate MPs | Instrument | LTQ-FT hybrid mass spectrometer (Thermo Fisher Scientific) | ||||
MP isolation | 2× Cent. 40,000 g/20 min/4 °C followed by Flow assisted cell sorting to isolate erythrocyte and platelet derived MPs. | Software for protein ID | Mascot v 2.1 | |||||
Protein database | NCBInr (Human) Taxonomy: Human # Entries: not stated | |||||||
Granulocyte-derived MPs | Dalli et al. [10] | Determine MP proteomic changes due to different granulocyte stimuli, identify candidate granulocyte-derived MP biomarkers, and test a panel of identified MP-biomarkers in sepsis patients and controls | Individuals: (a) 16 healthy donors (b) 10 donors with blister exudate (c) 10 donors with sepsis | Sample collection | Whole-blood Anticoagulant: not stated | Workflow | 1-D gel electrophoresis (Silver staining); bands of interest were out and in-gel digested MS analysis Differentially expressed proteins were identified by using normalized spectral counts | 342 proteins could be identified from MPs derived in vitro from neutrophils stimulated in immobilized phase (post adhesion to HUVECs) 302 proteins were identified from MPs derived in vitro from neutrophils stimulated in fluid phase 30% of the proteins were uniquely expressed in one of the two MP classes A2M and ceruloplasmin were particular enriched in the immobilized phase induced MPs, whereas heat shock 70 kDa protein 1 was enriched in fluid phase induced MPs, and Annexin A1 was expressed equally between the two classes of MPs Granulocyte-derived MPs from plasma showed—using flow cytometry—significantly different expression of these proteins between septic patients, healthy controls and exudate MPs |
Sex: (a) 9 males and 7 females (b) 6 males and 4 females (c) 2 males and 8 females | PRP generation (cell removal) | Cent. 1600 g/10 min/4 °C | Tryptic digestion | In-gel digestion | ||||
Age: (a) 30.0 ± 0.5 yrs (b) 31.3 ± 0.4 yrs (c) 58.8 ± 1.6 yrs | PPP/PFP generation (Plt removal) | Not performed | Method | Nano-LC–MS/MS | ||||
Ethnicity: not stated | Storage of PPP/PFP before MP isolation | PRP was stored at −80 °C before MP isolation | Instrument | LTQ-Orbitrap XL (Thermo Fisher Scientific) | ||||
MP isolation | 2× Cent. 3000 g/10 min/4 °C followed by Cent. 100,000 g/60 min/4 °C (Pellet = MPs) Isolated MPs were frozen at −80 °C before analysis | Software for protein ID | SEQUEST v28 in combination with X!Tandem v2007.01.01.2. In addition Scaffold v2.0.5 was used for peptide and protein validation and for spectral count calculation | |||||
In vitro generated granulocyte-derived MPs | Granulocytes were isolated from the buffy coat using Ficoll-Hypaque. Granulocytes were stimulated by fMLF activation (fluid phase) or incubation with a HUVEC monolayer (solid phase). MP isolation: centrifugation by 3000 g/10 min/4 °C twice, then 100,000 g/60 min/4 °C and frozen at −80 °C | Protein database | UniProtKB/SwissProt v14.6 Taxonomy: Human # Entries: 20,333 | |||||
Exudate MPs | Blisters were induced by cantharidin and exudates were harvested after 24 h, MP isolation not stated |