MassIVE MSV000100682

Partial Public PXD073879

Fractionated TMT32 bridge channel samples from healthy PBMC donor for generation of FAIMS RTLS spectral libraries

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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. [doi:10.25345/C5D795Q7X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: spectral library ; RTLS ; scProteomics ; single-cell proteomics ; PBMCs ; immune cells ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		James M. Fulcher, Pacific Northwest National Laboratory, USA
Ljiljana Pasa-Tolic, Pacific Northwest National Laboratory, USA
Submitting User: alchemistmatt
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