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

J Proteome Res. 2015 Feb 6;14(2):829-38. doi: 10.1021/pr500882h. Epub 2014 Dec 12.

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 prefractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a post-mortem brain sample of Alzheimer's disease. Because deep RNA sequencing of the same specimen suggested that ∼16,000 genes were expressed, the current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.

Keywords: AD proteome; long LC column; mass spectrometry.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease / metabolism
  • Animals
  • Brain / metabolism
  • Brain Chemistry / physiology*
  • Chromatography, Liquid / methods*
  • Humans
  • Proteome / analysis*
  • Proteomics / methods*
  • Rats
  • Tandem Mass Spectrometry / methods*

Substances

  • Proteome