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PXD073879
PXD073879 is an original dataset announced via ProteomeXchange.
Dataset Summary
| Title | Fractionated TMT32 bridge channel samples from healthy PBMC donor for generation of FAIMS RTLS spectral libraries |
| Description | This is the first of three MassIVE repositories associated with the manuscript "Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day." This repository contains datasets generated from a healthy human PBMC donor to create a spectral library for RTLS analysis. Bulk PBMC peptides were TMTpro 32-plex labeled (channels 126 and 135ND, pooled 1:1), microfractionated, and analyzed using FAIMS-enabled LC-MS/MS (Orbitrap and ion trap MS2) on a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer. [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Proteins were quantified by BCA, diluted, and sequentially digested with Lys-C and trypsin (1:50 enzyme-to-protein ratios). Peptides were desalted using C18 MacroSpin columns, concentrated, quantified, and dried prior to labeling. [TMTpro Labeling]: Peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 or 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL. [High-pH Microfractionation]: TMT-labeled peptides were separated by high-resolution reverse-phase capillary LC (nanoACQUITY UPLC) using a 200 um x 65 cm C18 column. Fractions were collected every minute from 65-170 min, concatenated in a staggered pattern, dried, and reconstituted in 2% acetonitrile/0.1% formic acid. [LC-MS/MS Analysis]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to an Orbitrap Eclipse Tribrid MS with FAIMS. Columns were operated at 100 nL/min with a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were used. Two spectral library acquisition methods were applied: 1) Orbitrap MS2: HCD fragmentation, 120k MS1 resolution, Orbitrap MS2 detection; 2) Ion Trap MS2: HCD fragmentation with rapid ion trap MS2 detection. [Database Searching and Spectral Library Generation]: Raw files were processed in FragPipe v23.0 using MSFragger, MSBooster, Percolator, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24) with 20 ppm precursor tolerance, tryptic/semi-tryptic specificity, static TMTpro modifications on lysine and N-termini, and methionine oxidation as a variable modification. Protein FDR was set to 1%.MS2 spectra were filtered to the top 150 fragment ions and converted to mzML. Spectral libraries were generated in SpectraST for each FAIMS CV and MS2 analyzer, converted to mzVault .db format, and merged to produce final Orbitrap and ion trap spectral libraries for -45 V and -65 V FAIMS conditions. |
| HostingRepository | MassIVE |
| AnnounceDate | 2026-02-01 |
| AnnouncementXML | Submission_2026-02-01_18:32:35.220.xml |
| DigitalObjectIdentifier | |
| ReviewLevel | Non peer-reviewed dataset |
| DatasetOrigin | Original dataset |
| RepositorySupport | Unsupported dataset by repository |
| PrimarySubmitter | Matthew Monroe |
| SpeciesList | scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606; |
| ModificationList | Acetyl; TMTpro |
| Instrument | Orbitrap Eclipse |
Dataset History
| Revision | Datetime | Status | ChangeLog Entry |
|---|---|---|---|
| 0 | 2026-01-31 16:26:29 | ID requested | |
| ⏵ 1 | 2026-02-01 18:32:35 | announced |
Publication List
| no publication |
Keyword List
| submitter keyword: spectral library, RTLS, scProteomics, single-cell proteomics, PBMCs, immune cells, DatasetType:Proteomics |
Contact List
| Ljiljana Pasa-Tolic | |
|---|---|
| contact affiliation | Pacific Northwest National Laboratory |
| contact email | ljiljana.pasatolic@pnnl.gov |
| lab head | |
| James M. Fulcher | |
| contact affiliation | Pacific Northwest National Laboratory |
| contact email | james.fulcher@pnnl.gov |
| lab head | |
| Matthew Monroe | |
| contact affiliation | Pacific Northwest National Laboratory |
| contact email | matthew.monroe@pnnl.gov |
| dataset submitter | |
Full Dataset Link List
| MassIVE dataset URI |
| Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://massive-ftp.ucsd.edu/v12/MSV000100682/ |
