Evaluation of Liquid Chromatography-Mass Spectrometry for Routine Proteome Analyses

Many different separation strategies have been developed for conducting proteomic studies, but microcapillary reversed-phase liquid chromatography (μLC) coupled online to mass spectrometry (MS) has played an undeniably central role. We have conducted a study to evaluate two different common column configurations, along with two common stationary phase materials, for their ability to identify peptides from a complex proteome digest. A 10 cm long x 75 μm inner diameter (id) capillary column with an integrated electrospray tip, and a 30 cm long × 75 μm id capillary column in which the electrospray tip is separated from the separation capillary via a stainless steel union, both packed in-house with reversed phase C₁₈, 5 μm, 300 Å pore size particles, were evaluated for their ability to effectively resolve a complex mixture of tryptic peptides from a mouse cortical neuron proteome sample for online tandem MS (MS/MS) analysis. The results demonstrate that the continuous 10 cm long x 75 μm id capillary column with the integrated electrospray tip, enables the identification of a comparable number of peptides in a single μLC-MS/MS analysis of a proteome tryptic digestate. We further evaluated the relationship between the numbers of peptides identified vs. the amount of sample loaded onto a 10cm long x 50 μm id column packed with 3 μm, 100 Å, C₁₈ reversed phase particles. The results show that more than 100 peptides can be identified from as little as 5 ng of tryptic peptides loaded, and that the number of identified peptides did not significantly increase beyond the loading of 50 ng.