Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome.

ABSTRACT: The development of high-resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse-phase long column (100 μm × 150 cm, 5 μm C18 beads) coupled to Q Exactive MS. The column was capable of achieving a peak capacity of ∼700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was ∼6 μg of peptides, although the column allowed loading as many as 20 μg. Gas-phase fractionation of peptide ions further increased the number of peptide identification by ∼10%. Moreover, the combination of basic pH LC prefractionatio

SUBMITTER: Wang H 

PROVIDER: S-EPMC4324436 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature