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s":null,"additional":{"labhead_mail":["MAIKAWA@BWH.HARVARD.EDU"],"submitter":["Sasha  Singh"],"technology_type":["Mass Spectrometry","Data-independent acquisition"],"software":[""],"submitter_keywords":["Biofluids","Clinical proteomics","Mass spectrometry","Data-independent acquisition"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD069212"],"tissue":["Blood Plasma"],"sample_protocol":["Human plasma samples were purchased from 5 independent commercial sources (Sources 1 to 5) - 2 male and 2 female donors each with ages equally distributed from 20 to 65 years. To reduce confounding results due to potential donor outliers, we pooled each sourced samples, and aliquot the pool into 4 reactions for each plasma preparation (4 ENRICHplus vs. 4 iST-BCT, more below).  To evaluate to what extent workflow operator impacted proteome yield or consistency, 12 aliquots of the Source 1 pool were made for the ENRICHplus workflow. Researchers TK, LW and JW performed 4 replicates each, whereas only JW performed the ENRICHplus workflow for the remaining source samples. These preparations were done the same day. Researcher TK performed the iST-BCT kit preparations using pooled aliquots generated at the time of initial sample preparation. Plasma proteome enrichment and proteolysis were performed using the ENRICHplus kit (PreOmics, GmbH, Germany). For each source, the 4 plasma samples were pooled and 4 aliquots of 50 µL for Sources 2 to 5, and 12 aliquots for Source 1. The 50 µL aliquot was used as in the input for the ENplus-BEADS. The remaining steps of the protocol were according to the manufacturer’s instruction. The eluted peptides were dried in a speed vacuum (Eppendorf Vacufuge) at 45 ˚C and resuspended in 22 µL LC-LOAD provided by the kit. For neat plasma proteomics, 2 µL of plasma was prepared using the iST-BCT kit according to manufacturer’s protocol (PreOmics, Germany). The dried, eluted peptides were resuspended in 42 µL LC-LOAD. The peptide amount was determined by using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). Each peptide sample was diluted to 100 ng/µL using LC-LOAD before injection into the LC-MS/MS."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The Exploris 480 spectra were analyzed using Proteome Discoverer (PD, version 3.2 with CHIMERYS, Thermo Fisher Scientific) or DIA-NN version 2.0 Enterprise. The timsTOF HT spectra were analyzed using DIA-NN 2.0 Enterprise. PD3.2+CHIMERYS - The spectra were queried against the Uniprot human database (downloaded May 2025; 42,421 entries). Trypsin/P was set as the digestion enzyme, allowing up to 2 missed cleavages and a minimum peptide length of 7-30 amino acids (1-6 peptide charge). Oxidation (+15.995 Da) of methionine was set as a variable modification, and carbamidomethylation (+57.021 Da) of cysteine was set as a static modification. The Quantification Type was set to the default setting, MS2 Apex (Quan in all files). Three consensus proteomes were exported: 1) a consensus comprising 28 ENRICHplus spectral files (12 for Source 1, 4 for Sources 2 to 5); 2) single consensuses comprising the 4 replicate samples per source; and 3) a consensus combining the 20 iST-BCT spectral files. DIA-NN - The spectra were queried against the same Uniprot human database as above, in library-free mode. Trypsin/P was set as the digestion enzyme, allowing up to 1 missed cleavage and a minimum peptide length of 7-30 amino acids (1-4 peptide charge). N-terminal methionine excision and carbamidomethylation (+57.021 Da) of cysteine were considered. For the algorithm settings, match between runs and protein inference were selected. The quantification strategy was set to Quant UMS (high precision), and the cross-run normalization was retention time-dependent. For each Exploris 480 and timsTOF HT runs, two consensus proteomes were exported: 1) a consensus comprising 20 ENRICHplus spectral files (4 replicates per source); and 2) single consensuses comprising the 4 replicate samples per source. Our study reports proteins that are supported by a single peptide (1% FDR) that also had a quantification value in at least 1 of 4 replicates."],"omics_type":["Proteomics"],"labhead":["Masanori Aikawa"],"instrument_platform":[""],"submission_type":["COMPLETE"],"labhead_affiliation":["Yoshihiro Miwa Distinguished Chair & Founding Director,  Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women’s Hospital;  Professor of Medicine, Harvard Medical School"],"species":["Homo Sapiens (human)"],"publication":["41879802 Kasai T, Weiss L, Wohlfahrt J, Matamalas JT, Shlayen G, Kirsch ZJ, Aikawa M, Aikawa E, Singh SA. Impact of Variations in Commercial Standard Operating Procedure on Plasma Proteome Recovery. J Proteome Res. 2026 25(4):1941-1950 10.1021/acs.jproteome.5c01012"],"submitter_mail":["sasingh@bwh.harvard.edu"],"submitter_affiliation":["Brigham and Women's Hospital, Harvard Medical School"],"submitter_country":["United States"],"doi":["10.6019/PXD069212"],"pubmed_abstract":["Advancements in mass spectrometry and complementary technologies now enable comprehensive, high-resolution plasma proteomics. Plasma is a key biofluid for clinical research, harboring potential disease-informative biomarkers. Commercial sources of nondiseased, healthy donor plasma samples are often used for proteomic workflow development and as controls for clinical studies. The overarching assumption is that standard operating procedures for plasma preparation are comparable across different sources. In this study, we investigated the effectiveness of a particle-based protein enrichment strategy against a conventional proteomics workflow on plasma samples from five commonly used commercial sources. We aimed to characterize the extent of variability in plasma proteomes when factors such as freeze-thaw cycles, choice of anticoagulant, and operator-to-operator performance were accounted for in the study setup. Plasma samples were analyzed in data-independent acquisition mode by using two distinct instruments (Exploris 480, timsTOF HT) and search algorithms (CHIMERYS, DIA-NN). Plasma proteome enrichment yields ranged from 2.8- to 6.2-fold more compared to the conventional workflow, achieving yields exceeding 5000 proteins (timsTOF HT). Notably, the observed variability in proteome composition was largely attributable to differences in whole blood-to-plasma operating procedures across commercial sources. While these distinct proteomes remained undetectable with conventional workflows, particle-dependent proteome profiling successfully revealed the procedural differences."],"pubmed_title":["Impact of Variations in Commercial Standard Operating Procedure on Plasma Proteome Recovery."],"pubmed_authors":["Kasai Taku T, Weiss Luisa L, Wohlfahrt Jessica J, Matamalas Joan T JT, Shlayen Gabriel G, Kirsch Zachary J ZJ, Aikawa Masanori M, Aikawa Elena E, Singh Sasha A SA"],"additional_accession":[]},"is_claimable":false,"name":"Impact of variations in standard operating procedure on plasma proteome recovery","description":"Advancements in mass spectrometry and complementary technologies now enable comprehensive and high-resolution plasma proteomics. Plasma is a key biofluid for clinical research, harboring potential disease informative biomarkers. Commercial sources of non-diseased, healthy donor plasma samples are often used for proteomic workflow development and as controls for clinical studies. The overarching assumption is that standard operating procedures for plasma preparations are comparable across variable sources. In this study, we investigated the effectiveness of a particle-based protein enrichment strategy against a conventional proteomics workflow on plasma samples from 5 commonly used sources aiming to characterize the extent of variability when factors such as freeze-thaw cycles, anticoagulant, and operator-to-operator performance were controlled.  Plasma samples were analyzed in data-independent acquisition mode using two distinct instruments (Exploris 480, timsTOF HT) and search algorithms (CHIMERYS, DIA-NN). Plasma proteome enrichment yields ranged from 4.5 to 8-fold more compared to the conventional workflow, achieving yields exceeding 5,000 proteins.  Notably, the observer variability in proteome composition was largely attributable to differences in whole blood-to-plasma operating procedures across the sources. While these distinct proteomes remained undetectable with conventional workflows, particle-dependent proteome profiling successfully revealed the procedural differences.","dates":{"publication":"2026-04-13","submission":"2025-10-08"},"accession":"PXD069212","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:10804","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:6493","NEWT:10832","NEWT:6494","NEWT: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