Nano-flow LC-MS has been the primary approach due to its high sensitivity. However, challenges always come from the needs of high throughput, reproducibility and robustness. Here we present a micro-flow LC-MS workflow using a robust setup with Thermo Fisher ScientificTM NG micro-flow UHPLC System coupled to Thermo Fisher ScientificTM Orbitrap ExplorisTM 240 mass spectrometer. Gas-phase fractionation (GPF) was performed to improve protein and peptide coverage using Thermo Fisher ScientificTM High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Pro Duo interface. We further applied this platform to scale up the characterization of proteomes using peripheral blood mononuclear cells (PBMCs) across different organisms.
Label-free proteomics performance on the micro-flow LC-MS setup was evaluated at 3 different loads of HeLa protein digest, 1ug, 5ug, 10ug, separated at both 30min and 50min gradient lengths on a 2 µm, 15 cm PepMap column. Each experiment condition was repeated for 5 injections in order to demonstrate the reproducibility. We were able to identify ~3400 protein groups and ~27500 peptide groups from 1ug of HeLa digest, ~3800 protein groups and ~34000 peptide groups from 5ug of HeLa digest within 30min gradient. The micro-flow LC-MS system showed excellent reproducibility of protein group IDs (<3% coefficient of variation, CV) and protein group abundance (median CV <11%). Multiplex quantitation analysis was carried out on the same setup introduced above, by injecting 5ug, 10ug and 20ug TMT 11plex Yeast digest. On average of 5 replicates, 90% of identified proteins and peptides were successfully quantified. Peripheral blood mononuclear cells (PBMCs) from a variety of animal species (human, mouse, rat, non-human primate, canine, and minipig) were purchased from BioIVT, followed by cell lysate and protein digestion. FAIMS Pro Duo interface was coupled to micro-flow LC to offer further separations in gas phase. Digested peptides from each animal species were fractioned by 6 compensation voltages and were acquired for triplicates on Orbitrap Exploris 240 MS. GPF would provide a deep proteome profiling without the need for off-line RPLC fractionation, which largely saved the overall experiment time. This micro-flow LC-MS setup has been demonstrated to be highly reproducible and robust without sacrificing the outstanding performance in proteomics applications for both discovery and quantitation.