DOI: 10.25345/c58g8fx3s
title: MassIVE MSV000100683 - Optimization of dual-column LC system and comparison of RTLS vs standard MS2 approach via healthy donor PBMC samples
title: This is the second 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 TMTpro 32-plex-labeled datasets from a healthy human PBMC donor, including both bulk (10 ng) and single-cell proteomic measurements. [PBMC Isolation, Digestion, and Cleanup]: Cryopreserved PBMCs were thawed, washed, and lysed in 8 M urea (50 mM TEAB). Protein concentration was measured 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 prior to 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 in nanoliter volumes using TMT-compatible buffer containing Lys-C and trypsin and incubated overnight at 37 deg C. TMTpro 32-plex reagents were dispensed directly onto 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]: 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. Two acquisition strategies were used: 1) Standard MS2 Method: Orbitrap MS2 detection following HCD fragmentation; 2) RTLS Method: Initial ion-trap MS2 scans were searched in real time against FAIMS-specific spectral libraries, triggering Orbitrap MS2 acquisition for matched precursors. Library matching used a 20 ppm precursor tolerance and cosine score &gt;= 40. [Database Searching and Quantification]: 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). For RTLS datasets, 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%. TMT quantification was performed using IonQuant and TMT-Integrator with median aggregation and MS1-based ratio-to-abundance conversion.