{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T15:27:10Z","timestamp":1788535630725,"version":"build-2803163510"},"reference-count":57,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R35 GM156406"],"award-info":[{"award-number":["R35 GM156406"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Nanoflow liquid chromatography coupled with tandem mass spectrometry (LC\u2013MS\/MS) underpins modern quantitative proteomics, yet the column-to-mass spectrometer interface remains an important yet often underappreciated determinant of analytical depth, sensitivity, and reproducibility. Here, we benchmark an integrated workflow comprising the newly developed OptiSpray ion source and a micropillar array column (\u03bcPAC) cartridge against a conventional Nanospray Flex Source with an Accucore resin-packed capillary column. We performed a TMTpro 18-plex experiment across nine human cell lines on a FAIMS Pro-equipped Orbitrap Exploris 480. Following basic-pH reversed-phase fractionation, 12 fractions were analyzed on both workflow configurations under matched chromatographic gradient and acquisition conditions. Across both configurations, we quantified &amp;gt;9000 protein groups with highly comparable quantitative reproducibility and principal component clustering. Direct comparison of protein abundance ratios across cell lines showed agreement (Pearson R2 \u2248 0.7\u20130.8) without systematic bias. These results were achieved without workflow-specific optimization of the OptiSpray\u2212\u03bcPAC platform, enabling direct transfer of established acquisition methods. Despite differences in column architecture, both configurations delivered comparable proteome coverage and quantitative fidelity. These findings establish the OptiSpray\u2212\u03bcPAC workflow as a standardized alternative to conventional capillary-based interfaces, offering simplified operation while preserving quantitative performance.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00473","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T06:16:35Z","timestamp":1788243395000},"page":"4987-4999","source":"Crossref","is-referenced-by-count":0,"title":["Benchmarking\nthe\nOptiSpray\u2212\u03bcPAC Workflow\nagainst a Traditional Nanospray Capillary Interface for Multiplexed\nQuantitative Proteomics"],"prefix":"10.1021","volume":"25","author":[{"given":"Katherine L.","family":"Walker","sequence":"first","affiliation":[{"name":"Thermo Fisher Scientific , , ,","place":["San Jose, California, United States, 95134"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina B.","family":"Schroeter","sequence":"additional","affiliation":[{"name":"Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"University Hospital Muenster , , ,","place":["Muenster, Germany, 48149"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5433-2540","authenticated-orcid":true,"given":"Runsheng","family":"Zheng","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific , , ,","place":["San Jose, California, United States, 95134"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua A.","family":"Silveira","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific , , ,","place":["San Jose, California, United States, 95134"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eloy R.","family":"Wouters","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific , , ,","place":["San Jose, California, United States, 95134"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4291-413X","authenticated-orcid":true,"given":"Joao A.","family":"Paulo","sequence":"additional","affiliation":[{"name":"Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"issue":"16","key":"2026090409135740400_cit1","doi-asserted-by":"publisher","first-page":"4235","DOI":"10.1021\/ac0202280","article-title":"High-efficiency nanoscale liquid chromatography coupled\non-line with mass spectrometry using nanoelectrospray ionization for\nproteomics","volume":"74","author":"Shen","year":"2002","journal-title":"Anal. Chem."},{"issue":"4","key":"2026090409135740400_cit2","doi-asserted-by":"publisher","first-page":"399","DOI":"10.1038\/s41592-020-0781-4","article-title":"TMTpro reagents:\na set of isobaric labeling mass tags enables simultaneous proteome-wide\nmeasurements across 16 samples","volume":"17","author":"Li","year":"2020","journal-title":"Nat. Methods"},{"issue":"8","key":"2026090409135740400_cit3","doi-asserted-by":"publisher","first-page":"1895","DOI":"10.1021\/ac0262560","article-title":"Tandem mass tags: a novel quantification\nstrategy for comparative analysis of complex protein mixtures by MS\/MS","volume":"75","author":"Thompson","year":"2003","journal-title":"Anal. Chem."},{"issue":"6","key":"2026090409135740400_cit4","doi-asserted-by":"publisher","first-page":"2226","DOI":"10.1021\/acs.jproteome.8b00217","article-title":"Streamlined Tandem Mass Tag (SL-TMT)\nProtocol: An Efficient Strategy for Quantitative (Phospho)proteome\nProfiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3","volume":"17","author":"Navarrete-Perea","year":"2018","journal-title":"J. Proteome Res."},{"issue":"1\u20132","key":"2026090409135740400_cit5","doi-asserted-by":"publisher","first-page":"233","DOI":"10.1016\/S0021-9673(02)01402-4","article-title":"Microcolumns with self-assembled\nparticle frits for proteomics","volume":"979","author":"Ishihama","year":"2002","journal-title":"J. Chromatogr\nA"},{"key":"2026090409135740400_cit6","doi-asserted-by":"publisher","first-page":"111","DOI":"10.1016\/j.chroma.2017.03.043","article-title":"Self-packed\ncore shell nano liquid chromatography columns and silica-based monolithic\ntrap columns for targeted proteomics","volume":"1498","author":"Berg","year":"2017","journal-title":"J. Chromatogr\nA"},{"issue":"3","key":"2026090409135740400_cit7","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1016\/j.jasms.2008.10.021","article-title":"A method for monitoring and controlling\nreproducibility of intensity data in complex electrospray mass spectra:\na thermometer ion-based strategy","volume":"20","author":"Lecchi","year":"2009","journal-title":"J. Am. Soc.\nMass Spectrom."},{"issue":"5","key":"2026090409135740400_cit8","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1021\/jasms.0c00052","article-title":"A Plug-and-Play\nHigh-Pressure ESI Source with an Emitter at Ground Potential and Its\nApplication to High-Temperature Capillary LC-MS","volume":"31","author":"Chen","year":"2020","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"4\u20136","key":"2026090409135740400_cit9","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1080\/14789450.2023.2219844","article-title":"Evaluation\nof a \u2019plug and\nplay\u2019 nanoflow liquid chromatography system for MS-based proteomic\ncharacterization of clinical FFPE specimens","volume":"20","author":"Mitsa","year":"2023","journal-title":"Expert Rev. 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Chem."},{"issue":"1","key":"2026090409135740400_cit12","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-45391-z","article-title":"Micropillar arrays,\nwide window acquisition and AI-based data analysis improve comprehensiveness\nin multiple proteomic applications","volume":"15","author":"Matzinger","year":"2024","journal-title":"Nat. Commun."},{"key":"2026090409135740400_cit13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.aca.2019.08.068","article-title":"Multidimensional performance assessment\nof micro pillar array column chromatography combined to ion mobility-mass\nspectrometry for proteome research","volume":"1086","author":"Nys","year":"2019","journal-title":"Anal. 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Chem."},{"issue":"9","key":"2026090409135740400_cit16","doi-asserted-by":"publisher","first-page":"4845","DOI":"10.1021\/acs.jproteome.5c00465","article-title":"Comparative\nProteomic Analysis of\nCapillary and Micropillar Array Columns Using DIA: Insights from the\nTKO Yeast Standard","volume":"24","author":"Zhang","year":"2025","journal-title":"J. Proteome Res."},{"issue":"5","key":"2026090409135740400_cit17","doi-asserted-by":"publisher","first-page":"701","DOI":"10.1038\/nprot.2015.040","article-title":"One-hour proteome analysis in yeast","volume":"10","author":"Richards","year":"2015","journal-title":"Nat. Protoc."},{"issue":"4","key":"2026090409135740400_cit18","doi-asserted-by":"publisher","first-page":"716","DOI":"10.1074\/mcp.TIR119.001906","article-title":"A Compact Quadrupole-Orbitrap Mass Spectrometer with\nFAIMS Interface Improves Proteome Coverage in Short LC Gradients","volume":"19","author":"Bekker-Jensen","year":"2020","journal-title":"Mol. Cell Proteomics"},{"issue":"15","key":"2026090409135740400_cit19","doi-asserted-by":"publisher","first-page":"9529","DOI":"10.1021\/acs.analchem.8b02233","article-title":"Comprehensive Single-Shot\nProteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer","volume":"90","author":"Hebert","year":"2018","journal-title":"Anal. Chem."},{"issue":"6","key":"2026090409135740400_cit20","doi-asserted-by":"publisher","first-page":"4010","DOI":"10.1021\/acs.analchem.8b05399","article-title":"Characterization\nand Optimization of Multiplexed Quantitative Analyses Using High-Field\nAsymmetric-Waveform Ion Mobility Mass Spectrometry","volume":"91","author":"Schweppe","year":"2019","journal-title":"Anal. 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Biotechnol."},{"issue":"10","key":"2026090409135740400_cit24","doi-asserted-by":"publisher","first-page":"1285","DOI":"10.1038\/nbt1240","article-title":"A probability-based\napproach for high-throughput protein phosphorylation analysis and\nsite localization","volume":"24","author":"Beausoleil","year":"2006","journal-title":"Nat. Biotechnol."},{"issue":"7","key":"2026090409135740400_cit25","doi-asserted-by":"publisher","first-page":"1174","DOI":"10.1016\/j.cell.2010.12.001","article-title":"A tissue-specific\natlas of mouse protein phosphorylation\nand expression","volume":"143","author":"Huttlin","year":"2010","journal-title":"Cell"},{"key":"2026090409135740400_cit26","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1007\/978-1-60761-444-9_5","article-title":"Target-decoy search\nstrategy for mass spectrometry-based\nproteomics","volume":"604","author":"Elias","year":"2010","journal-title":"Methods Mol. Biol."},{"issue":"3","key":"2026090409135740400_cit27","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1038\/nmeth1019","article-title":"Target-decoy search\nstrategy for increased confidence\nin large-scale protein identifications by mass spectrometry","volume":"4","author":"Elias","year":"2007","journal-title":"Nat. Methods"},{"issue":"17","key":"2026090409135740400_cit28","doi-asserted-by":"publisher","first-page":"7469","DOI":"10.1021\/ac301572t","article-title":"Increasing the Multiplexing Capacity\nof TMTs Using Reporter Ion Isotopologues with Isobaric Masses","volume":"84","author":"McAlister","year":"2012","journal-title":"Anal. Chem."},{"issue":"1","key":"2026090409135740400_cit29","doi-asserted-by":"publisher","first-page":"4","DOI":"10.32614\/RJ-2015-001","article-title":"Peptides: A Package\nfor Data Mining of Antimicrobial Peptides","volume":"7","author":"Osorio","year":"2015","journal-title":"R\nJ."},{"issue":"1","key":"2026090409135740400_cit30","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1016\/0022-2836(82)90515-0","article-title":"A simple method for displaying the hydropathic character\nof a protein","volume":"157","author":"Kyte","year":"1982","journal-title":"J. Mol. Biol."},{"issue":"9\u201310","key":"2026090409135740400_cit31","doi-asserted-by":"publisher","first-page":"1737","DOI":"10.1002\/jssc.202000041","article-title":"Investigation of mass\ntransfer properties and kinetic\nperformance of high-efficiency columns packed with C18 sub-2 \u03bcm\nfully and superficially porous particles","volume":"43","author":"Felletti","year":"2020","journal-title":"J.\nSep. Sci."},{"issue":"45","key":"2026090409135740400_cit32","doi-asserted-by":"publisher","first-page":"8209","DOI":"10.1016\/j.chroma.2011.09.034","article-title":"On the relationship\nbetween band broadening and the particle-size distribution of the\npacking material in liquid chromatography: Theory and practice","volume":"1218","author":"Gritti","year":"2011","journal-title":"J. Chromatogr. A"},{"key":"2026090409135740400_cit33","doi-asserted-by":"crossref","unstructured":"De Beeck, J. O.; Pauwels, J.; Van Landuyt, N.; Jacobs, P.; De Malsche, W.; Desmet, G.; Argentini, A.; Staes, A.; Martens, L.; Impens, F.; Gevaert, K.\n          A well-ordered nanoflow LC-MS\/MS\napproach for proteome profiling using 200 cm long micro pillar array\ncolumns\n          bioRxiv\n          2019\n          10.1101\/472134.","DOI":"10.1101\/472134"},{"key":"2026090409135740400_cit34","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2020.461279","article-title":"Separation efficiency kinetics of\ncapillary flow micro-pillar array columns for liquid chromatography","volume":"1626","author":"Desmet","year":"2020","journal-title":"J. Chromatogr. A"},{"key":"2026090409135740400_cit35","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1016\/j.chroma.2011.12.088","article-title":"Estimation\nand optimization of the\npeak capacity of one-dimensional gradient high performance liquid\nchromatography using a long monolithic silica capillary column","volume":"1228","author":"Horie","year":"2012","journal-title":"J. Chromatogr. A"},{"issue":"7","key":"2026090409135740400_cit36","doi-asserted-by":"publisher","first-page":"1192","DOI":"10.1002\/jssc.201201087","article-title":"High-resolution\npeptide separations using nano-LC at ultra-high pressure","volume":"36","author":"Nova\u0301kova\u0301","year":"2013","journal-title":"J. Sep. Sci."},{"issue":"(7)","key":"2026090409135740400_cit37","doi-asserted-by":"publisher","first-page":"1060","DOI":"10.1002\/elps.201300327","article-title":"Comparison of CE-MS\/MS\nand LC-MS\/MS sequencing demonstrates significant complementarity in\nnatural peptide identification in human urine","volume":"35","author":"Klein","year":"2014","journal-title":"Electrophoresis"},{"key":"2026090409135740400_cit38","doi-asserted-by":"publisher","first-page":"10","DOI":"10.3390\/proteomes14010010","article-title":"Comparison of the Trapping Efficiency for Tryptic Peptides\non Particle-Packed and Micro-Pillar Trap Columns for Proteomics Analyses","volume":"14","author":"Vukajlovic\u0301","year":"2026","journal-title":"Proteomes"},{"key":"2026090409135740400_cit39","doi-asserted-by":"publisher","first-page":"5461","DOI":"10.1007\/s00216-022-03934-7","article-title":"Complementary\nProteome and Glycoproteome Access Revealed Through Comparative Analysis\nof Reversed Phase and Porous Graphitic Carbon Chromatography","volume":"414","author":"Delafield","year":"2022","journal-title":"Anal. Bioanal. Chem."},{"key":"2026090409135740400_cit40","doi-asserted-by":"publisher","first-page":"3508","DOI":"10.1021\/acs.jproteome.3c00373","article-title":"Inclusion\nof Porous\nGraphitic Carbon Chromatography Yields Greater Protein Identification,\nCompartment and Process Coverage, and Enables More Reflective Protein-Level\nLabel-Free Quantitation","volume":"22","author":"Delafield","year":"2023","journal-title":"J. Proteome Res."},{"issue":"46","key":"2026090409135740400_cit41","doi-asserted-by":"publisher","first-page":"15930","DOI":"10.1021\/acs.analchem.2c01196","article-title":"Deep Proteome\nProfiling\nwith Reduced Carryover Using Superficially Porous Microfabricated\nnanoLC Columns","volume":"94","author":"Stejskal","year":"2022","journal-title":"Anal. Chem."},{"issue":"2","key":"2026090409135740400_cit42","doi-asserted-by":"publisher","first-page":"761","DOI":"10.1021\/pr9006365","article-title":"Repeatability\nand Reproducibility\nin Proteomic Identifications by Liquid Chromatography\u2013Tandem\nMass Spectrometry","volume":"9","author":"Tabb","year":"2010","journal-title":"J. Proteome Res."},{"issue":"9\u201310","key":"2026090409135740400_cit43","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.202400187","article-title":"Data-Independent Acquisition Mass\nSpectrometry as a Tool for Metaproteomics: Interlaboratory Comparison\nUsing a Model Microbiome","volume":"25","author":"Rajczewski","year":"2025","journal-title":"Proteomics"},{"issue":"5","key":"2026090409135740400_cit44","doi-asserted-by":"publisher","first-page":"1400","DOI":"10.1074\/mcp.M114.044305","article-title":"Extending the limits of quantitative\nproteome profiling with data-independent acquisition and application\nto acetaminophen-treated three-dimensional liver microtissues","volume":"14","author":"Bruderer","year":"2015","journal-title":"Mol. Cell Proteomics"},{"issue":"1","key":"2026090409135740400_cit45","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1186\/s12014-025-09572-2","article-title":"In-depth analysis\nof data characteristics\nand comparative evaluation of dda and dia accuracy in label-free quantitative\nproteomics of biological samples","volume":"23","author":"Zou","year":"2026","journal-title":"Clin Proteomics"},{"key":"2026090409135740400_cit46","doi-asserted-by":"publisher","first-page":"10","DOI":"10.3390\/proteomes14010010","article-title":"Comparison of the Trapping Efficiency for Tryptic Peptides\non Particle-Packed and Micro-Pillar Trap Columns for Proteomics Analyses","volume":"14","author":"Miletic\u0301 Vukajlovic\u0301","year":"2026","journal-title":"Proteomes"},{"key":"2026090409135740400_cit47","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2024.465142","article-title":"Evaluating C18 stationary phases with a positively\ncharged surface for proteomic LC-MS applications using mobile phase\nacidified with reduced formic acid concentration","volume":"1730","author":"Jadeja","year":"2024","journal-title":"J. Chromatogr. A"},{"key":"2026090409135740400_cit48","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2024.464694","article-title":"Improved method robustness and ruggedness\nin liquid\nchromatography\u2013mass spectrometry by increasing the acid content\nof the mobile phase","volume":"1717","author":"Kaufmann","year":"2024","journal-title":"J. Chromatogr. A"},{"key":"2026090409135740400_cit49","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2020.461279","article-title":"Separation efficiency kinetics of\ncapillary flow micro-pillar array columns for liquid chromatography","volume":"1626","author":"Desmet","year":"2020","journal-title":"J. Chromatogr A"},{"issue":"1","key":"2026090409135740400_cit50","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1002\/jssc.201700863","article-title":"Optimizing\nseparations in online comprehensive two-dimensional liquid chromatography","volume":"41","author":"Pirok","year":"2018","journal-title":"J. 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