Conventional-Flow Liquid Chromatography–Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses

Abstract

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography−mass spectrometry (LC−MS) has become the core approach in the field. The de facto standard LC−MS platform for proteomics operates at sub-μL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC−MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC−MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC−MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 μg of HeLa cells tryptic digest separated during a 60 min gradient at 68 μL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua−acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC−MS is an attractive alternative for bottom-up exploratory proteomics.

Standard Flow Multiplexed Proteomics (SFloMPro) – An Accessible and Cost-Effective Alternative to NanoLC Workflows

“…5 The resulting methodology, NanoLC, was rapidly adopted in shotgun proteomics, to the point that it has been recently described as dogma. 6 NanoLC requires the precise plumbing of fragile fused silica columns with internal diameters ranging 20 -100 µm and flow rates typically ranging 20 -500 nL per minute. As few pumps existed that could reliably deliver these flow rates, early practitioners utilized solvent splits from higher flow pumps that would divert as much as 99% of the utilized solvent to waste with the remaining 1% used for peptide gradient elution.…”

“…Recent work has also described the use of "standard flow" proteomics, reaching the conclusion that flow rates in the 50 -200 µl per minute range can provide quality proteomics data with proper optimization. 6,18 Building on this work, we describe a standard flow multiplexed proteomics (SFloMPro) workflow. Our results show that SFloMPro produces comparable data to that of NanoLC but requires 20 times more labeled peptide on column.…”

“…All analyses used a Vanquish H uHPLC (Thermo) coupled to Q Exactive mass spectrometer. Starting conditions were based on a recent report by Lenco et al 6 using a Waters BEH Peptide BEH C18 1.7µm x 2.1 x 150mm column. Optimization of injection and gradient was performed using the HeLa peptide standard (Pierce).…”

“…An identical data-dependent acquisition method was used for both experiments. MS1 spectra were acquired at a resolution of 70,000 with an AGC target of 3x10 6…”

High-flow Nano-chromatography Columns Facilitate Rapid and High-quality Proteome Analysis on Standard Nano-LC Hardware

“…However, when coupled to MS, measurement sensitivity is not fully optimized for analytes with limited amounts of available material due to the large post-column elution volumes (sample dilution) and less efficient ionization. With significant optimization, these systems have been successfully applied to proteome analysis with MS [1][2][3][4] , but the required complexity in achieving these performance levels with this type of setup limits their widespread use.…”