Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

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 mu m X 150 cm, 5 mu m C18 beads) coupled to Q Exactive MS. The column was capable of achieving a peak capacity of similar to 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was similar to 6 mu g of peptides, although the column allowed loading as many as 20 mu g. Gas-phase fractionation of peptide ions further increased the number of peptide identification by similar to 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 similar to 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.