The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis

摘要

The microcapillary liquid chromatography (mLC)/tandem mass spectrometry (MS/MS) system hasnbecome a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteomewidenidentification of peptides and proteins rely heavily on producing an accurate and reproduciblensolvent-composition gradient throughout microcapillary separation columns to improve LCnseparation. With the recent advent of targeted proteomic approaches utilizing the LC retention time asna physicochemical parameter for peptides, high reproducibility of LC separation additionally becomesnan important factor. In this study, column temperature elevation is utilized to improve reproducibilitynand separation efficiency of the mLC-MS/MS system. The simple incorporation of a semi-rigid gas linenheater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up ton60 u0002C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlledntemperature elevation on the peptide separation efficiency and reproducibility. In addition to thenincreased reproducibility in peptide elution time due to the controlled column temperature, thentemperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowedna higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements innthe performance of mLC systems.