DOI: 10.25345/c54q7r39f
title: MassIVE MSV000100684 - Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day
title: This is the third 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 2,130 single PBMC measurements (71 raw files) acquired using a dual-column LC system with real-time library searching (RTLS). [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs from a healthy donor were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Protein concentration was determined by BCA, followed by dilution and sequential digestion 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 of Bulk Bridge Sample]: Bulk PBMC peptides were reconstituted in 50 mM bicine (pH 8.5). Two 10 ug aliquots were labeled with TMTpro channels 126 and 135ND, quenched with hydroxylamine, pooled, and diluted to 0.2 ug/uL for use as a bridge sample. [Single-Cell Isolation (cellenONE)]: PBMCs were thawed, washed, and resuspended in PBS before single-cell isolation using a cellenONE instrument. Cells were selected based on size, circularity, and elongation to exclude debris, apoptotic cells, and doublets. Individual cells were dispensed into 30 wells of 32-plex N2 nanoPOTS chips, with two wells reserved for bridge sample addition. Chips were stored at -80 deg C until processing. [N2 nanoPOTS Single-Cell Sample Preparation]: Single cells were digested overnight at 37 deg C in nanoliter volumes using TMT-compatible buffer containing Lys-C and trypsin. TMTpro 32-plex reagents were dispensed directly into each well using the cellenONE. Labeling proceeded overnight, followed by quenching with hydroxylamine, acidification with formic acid, evaporation, and storage at -20 deg C. TMTpro-labeled bridge sample was added at levels described in the manuscript. [LC-MS/MS Analysis with RTLS]: Samples were analyzed using a homebuilt dual-column nanoPOTS autosampler coupled to a ThermoFisher Orbitrap Eclipse Tribrid mass spectrometer with FAIMS. Dual parallel columns enabled near-continuous MS operation. Peptides were separated at 100 nL/min using a 75-min gradient. FAIMS compensation voltages of -45 and -65 V were applied. RTLS was implemented using rapid ion-trap MS2 scans searched in real time against FAIMS-specific spectral libraries. Precursors passing a cosine similarity threshold &gt;= 40 triggered high-resolution Orbitrap MS2 acquisition. Orbitrap MS1 scans were acquired at 120,000 resolution, followed by HCD fragmentation and Orbitrap MS2 detection for RTLS-selected precursors. [Database Searching and Quantification]: All raw files were processed using FragPipe v23.0 with MSFragger, MSBooster, Percolator, Philosopher, IonQuant, and TMT-Integrator. Searches used the UniProt Homo sapiens database (FASTA 08/16/24). Only high-resolution Orbitrap scans were retained for database searching. Search parameters included 20 ppm precursor tolerance, semi-tryptic specificity, static TMTpro modification on lysine and peptide N-termini, and methionine oxidation as a variable modification. Protein-level FDR was set to 1%. TMTpro quantification was performed using IonQuant and TMT-Integrator with median aggregation and MS1-based ratio-to-abundance conversion.