Nanoflow Low Pressure High Peak Capacity Single Dimension LC-MS/MS Platform for High-Throughput, In-Depth Analysis of Mammalian Proteomes

Abstract

The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600 minute gradient, with average chromatographic peak widths of less than one minute. At room temperature a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤ 10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.

Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

“…For additional coverage of the phosphoproteome, these phosphopeptides were analyzed on a higher-performance Orbitrap Fusion mass spectrometer 23,37 coupled to an improved version of our multidimensional fractionation platform. 24,38,39 In these analyses, we identified 12 323 and 12 168 unique phosphopeptide sequences present in all samples, representing 4001 and 3997 distinct phosphoproteins (supplemental Table 1; "first analysis" and "second analysis"). Across all 3 sets, 15 230 unique phosphopeptides and 4993 phosphoproteins were identified.…”

Protein Analysis by Shotgun/Bottom-up Proteomics

“…Reducing the column inner diameter and eluent flow to low nanoliter per minute rates while increasing the column length has produced similar results to longer multidimensional separations through both increased peak capacity and ionization efficiency. 183 High temperature RPLC has proven essential for the separation and identification of hydrophobic peptides from membrane-embedded proteins. 184 Sub-zero RPLC reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange experiments.…”

Differential contribution of the mitochondrial translation pathway to the survival of diffuse large B-cell lymphoma subsets

“…Our previous assessment of the mitochondrial proteome in OxPhos-and non-OxPhos/BCRdependent DLBCLs revealed the enrichment of several ETC subunits and ETC assembly factors in OxPhos-DLBCLs that is consistent with increased ETC activity in this subtype. 7 These previous analyses, based on a high-performance, single-dimension liquid chromatography-tandem mass spectrometry platform, 24 quantified predominantly nuclearencoded ETC subunits. Because OxPhos-DLBCLs display increased activity of several ETC complexes that are encoded by both the nuclear and mitochondrial genomes, 7 we predicted that the protein-level enrichment of ETC subunits in this subtype would also include mtDNAencoded subunits.…”