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

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

Author information

1
Department of Structural Biology, ‡St. Jude Proteomics Facility, and §̂Department of Developmental Neurobiology, St. Jude Children's Research Hospital , 262 Danny Thomas Place, Memphis, Tennessee 38105, United States.

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

PMID:
25455107
PMCID:
PMC4324436
DOI:
10.1021/pr500882h
[Indexed for MEDLINE]
Free PMC Article

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