{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/HYE.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/LL2112_NC_24hr_PBS100Gate_MBRDo.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/250pgHela.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/LL2112_Astral_Rawl_fullExpSeclib.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/LL2106_HelaWetDrySC_all_noMBR.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/RAW.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/LL2106_Bubis_libfree_noMBR.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/LL2106_HYE250pg_All_noDonor_noMBR.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD081782/HelaSC.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["ryan.kelly@byu.edu"],"submitter":["Ryan Kelly"],"technology_type":["Mass Spectrometry","Data-independent acquisition","Bottom-up proteomics"],"software":[""],"submitter_keywords":["High-throughput","Single-cell proteomics","Multicolumn nanolc"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD081782"],"tissue":["Macrophage"],"sample_protocol":["Cell culture and treatment: HeLa cells were cultured in 10-cm tissue culture plates using 10 mL of 1x DMEM (Corning, 10-013-CV). Media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All plates were incubated at 37 °C in a humidified incubator with 5% CO₂. Once HeLa cells reached confluency, they were rinsed with 5 mL of 1× phosphate-buffered saline (PBS), detached using 4 mL of 0.25% trypsin-EDTA (Gibco, 25200-072), and incubated at 37°C for 4 min. Trypsin activity was quenched by the addition of 10 mL of complete medium.  RAW264.7 cells were cultured in 10-cm plates using complete Corning DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 2 mM Gibco GlutaMAX. When cells reached approximately 60–70% confluency, they were washed once with PBS, and fresh complete medium was added. Lipopolysaccharide (LPS; Escherichia coli O111:B4, Sigma-Aldrich, L5293) was added to achieve a final concentration of 100 ng/mL by adding 50 µL of a 20 μg/mL LPS stock solution. For PBS-treated samples (control), an equivalent volume (50 µL) of PBS was added instead of LPS. All plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for 24 h, then collected as described for HeLa cells above.  Cell wash: All cell types were pelleted separately by centrifugation at 200 ×g for 10 min, resuspended in 2 mL of 1× PBS, and passed through a 40 µm filter. The cells were washed further by subsequent repelleting at 200 ×g for 5 min, resuspending in 1 mL of 1× PBS and repeating these steps to remove residual media and enzymes. Cell viability and diameter were assessed using Trypan Blue Stain (Gibco, Ref# 15250-061) and the Logos Luna II Automated Cell Counter. All cell types were adjusted to a final concentration of 2 × 10⁵ cells/mL before isolation.  Single-cell isolation: The single-cell sample preparation followed the protocol published previously67-69. In brief, single cells were dispensed into 0.01% DDM-treated 384-well PCR plates using the Tecan UNO dispenser (Männedorf, Switzerland). During dispensing, size-based gating was applied with a diameter range of 15–17 µm for HeLa cells and 9-11 µm for control RAW264.7 cells. For LPS-treated RAW 264.7 cells, a 9–11 µm and 12–14 µm diameter range was used for isolation. HeLa cells were dispensed onto a separate set of 384-well plates from the RAW 264.7 experiments. For RAW 264.7 cells, either control or LPS-treated cells were isolated onto the same plates, or LPS-treated cells from different diameter ranges were isolated onto separate plates. Plates were centrifuged at 380 ×g immediately following dispensing to ensure cells were driven to the bottom of each well. Single cells were randomized across conditions and analyzed to minimize batch effects over an extended period.  Digestion: A one-pot workflow was applied to SCP preparation to avoid sample loss during multiple transfer steps67-71. Immediately after isolation of all cell types, wells containing single cells were subjected to in-well proteolytic digestion69. A 200 nL volume of digest solution was dispensed into each well using the Tecan UNO dispenser. Each aliquot consisted of 10 nL of 0.2 µg/µL Rapid Trypsin/Lys-C (Promega, Cat# V507C) in Resuspension Buffer (Promega, Cat# V181A), 10 nL of 1% DDM in Rapid Digest Buffer (Promega, Cat# VA106A) and 180 nL of Rapid Digest Buffer, resulting in final concentrations of 10 ng/µL Trypsin/Lys-C and 0.05% DDM.  Plates were centrifuged, sealed with a silicone mat, wrapped in aluminum foil and incubated in a humidified oven at 70 °C for 1 h. After digestion, plates were cooled to 4 °C for 15 min, sealed with aluminum sealing foil, and stored at −20 °C until LC-MS/MS analysis. HeLa digest standard was prepared using Pierce™ HeLa Protein Digest Standard (ThermoFisher, Ref# 88329). Dilutions were prepared at 10 ng/µL for experimental spectral library creation, and at 250 pg/µL with 0.1% formic acid for system evaluation. The HeLa Digest standard was also prepared at 1.25 ng/µL and dispensed with the D100 HP dispenser at 200 nL per well to DDM-treated 384-well plates for the evaluation of sample recovery following reconstitution.  HYE digest standard: The HYE mixture was prepared using the same HeLa digest standard, yeast protein extract (Promega, Cat# V7461), and MassPREP™ E. coli Digest Standard (Waters, Part#186003196). Two mixtures were prepared: HYE A and HYE B. For HYE A, 100 µL each of the three components: HeLa, yeast, and E. coli digests were combined, yielding final concentrations of 20 ng/µL, 9.23 ng/µL, and 1.54 ng/µL, respectively. HYE B was prepared similarly, with final respective concentrations of 20 ng/µL, 4.62 ng/µL, and 6.15 ng/µL. The solution was diluted to achieve a final HeLa component concentration of 10 ng/µL for the creation experimental spectral library and 250 pg/µL for system evaluation."],"repository":["Pride"],"modification":[""],"quantification_method":["Not available"],"data_protocol":["Near-real-time monitoring: Individual MS runs were searched using DIA-NN 2.2.0 Academia through MSConnect72 with a Human UniProt database downloaded in December 2024 for both HeLa standard and single cell dataset; human, yeast, and E. coli UniProt protein databases downloaded in December 2024 were combined into a single search database for the HYE dataset; and the Mouse UniProt database downloaded in March 2026 was used for the RAW264.7 dataset. An experimental spectral library and three Proteomic Data Processors were employed to match the search time to a 5-minute LC-MS cycle time for near-real-time monitoring.  The experimental spectral libraries were created using 10 ng peptide standards or bulk-digested RAW 264.7 cells, which were analyzed on the same LC-MS system as the sample runs with the corresponding predicted spectral library. The following parameters were the same for predicted spectral library creation, experimental spectral library creation, and the data search. Trypsin/P was specified as the digestion enzyme, with a peptide length range of 7–54 amino acids and up to three missed cleavages. A maximum of three modifications were allowed per peptide, including N-terminal methionine excision, carbamidomethylation, N-terminal acetylation and methionine oxidation. The false discovery rate for the precursor was controlled at 1% using DIA-NN. Quantification was performed by DIA-NN’s default at the MS2 level, and cross-run normalization was disabled.  Identification and Quantification: While near-real-time monitoring was performed during acquisition using the workflow described above, all reported identifications and quantifications in this study were generated from a final DIA-NN search performed after completion of each experiment. In this final search, all MS runs from the same experiment were analyzed together against the corresponding experimental spectral library using DIA-NN 2.2.0 Academia, with the same search parameters as those used for near-real-time monitoring. For the column-to-column comparison (Figure 3), single-cell HeLa analysis (Figure 4) and HYE mixed-species standards (Figure 5), searches were performed without MBR and without inclusion of the 10 ng standard runs to preserve direct assessment of run-to-run system variation. In contrast, the RAW264.7 dataset (Figures 6–7) was searched with MBR and 10 ng bulk-digested RAW264.7 reference runs to maximize proteome coverage for biological interpretation."],"omics_type":["Proteomics"],"labhead":["Ryan T. Kelly"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States"],"species":["Homo Sapiens (human)","Escherichia Coli","Candida Albicans (yeast)","Mus Musculus (mouse)"],"publication":["Not available"],"submitter_mail":["ryan.kelly@byu.edu"],"submitter_affiliation":["Brigham Young University"],"submitter_country":["United States"],"additional_accession":[]},"is_claimable":false,"name":"High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time","description":"Mass spectrometry (MS)-based single-cell proteomics (SCP) enables proteome-wide analysis at single-cell resolution, offering insights into cellular heterogeneity, biological processes, and disease mechanisms. However, conventional nanoLC-MS workflows are constrained by long gradients and low throughput, limiting their application to small cohorts. Here, we present a multicolumn nanoLC–MS platform that achieves 5-minute separation windows with 100% duty cycles at ~100 nL/min, enabling the analysis of up to 288 single cells per day with minimal additional hardware. The system provides stable peptide separation, negligible carryover, and robust retention-time reproducibility across 1,200 consecutive injections. Over the course of this study, we successfully analyzed more than 4,000 samples at nearly 288 samples per day (SPD) throughput. The platform identified ~5,000proteins per 250 pg digest injection and an average of ~4,000 proteins per single HeLa cell with maxima exceeding 5,100, which is comparable to state-of-the-art longer-gradient workflows. Quantitative benchmarking with mixed-species standards confirmed the quantitative accuracy is largely maintained during extended acquisition . The workflow profiled 783 individual RAW264.7 macrophages cells exposed to LPS, enabling an unprecedented characterization of cellular heterogeneity within a uniform inflammatory stimulus. . Together, these results establish a robust and scalable platform for high-throughput SCP, demonstrating the feasibility of thousands of single-cell analyses within a single study while maintaining deep proteome coverage and biological interpretability.","dates":{"publication":"2026-08-19","submission":"2026-07-28"},"accession":"PXD081782","cross_references":{"TAXONOMY":["NEWT:68239","NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2737160","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:156563","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:93217","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:82569","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:1176302","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:77151","NEWT:317447","NEWT:5537","NEWT:698761","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:1177","NEWT:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:3134131","NEWT:49928","NEWT:160621","NEWT:110368","NCBITaxon:5204","NEWT:1916073","NEWT:1303873","NEWT:1310161","NEWT:1230383","NEWT:77133","NCBITaxon:5207","NEWT:160620","NCBITaxon:77643","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:1410384","NEWT:1310173","NEWT:37919","NEWT:1072569","NEWT:1072568","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1336794","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3710","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:284811","NEWT:980415","NEWT:2902907","NEWT:3701","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:10882","NEWT:1431297","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:34878","NEWT:10886","NEWT:3707","NEWT:3708","NEWT:189423","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:319224","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:223074","NEWT:2709072","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:387344","NEWT:657318","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:1385720","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2422","NEWT:2423","NEWT:3755","NEWT:133894","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:123606","NEWT:85698","NEWT:291146","NEWT:29466","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:34839","NEWT:476","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NEWT:106299","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:64697","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:1120755","NEWT:1862567","NEWT:591090","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","NEWT:1431340","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:52694","NEWT:554","NEWT:2642239","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:366497","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:771875","NEWT:377142","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:595495","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:6055","NEWT:13037","NEWT:518","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330"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