Evaluation of Liquid Chromatography–Mass Spectrometry for Routine Proteome Analyses
Journal of Liquid Chromatography & Related Technologies
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 × 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 × 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 10 cm long × 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.
Proteomites, liquid chromatography, mass spectrometry, peptides
Yu, Li-Rong; Issaq, Haleem J.; Conrads, Thomas P.; Uo, Takuma; Blonder, Josip; Janini, George m.; Morrison, Richard S.; and Veenstra, Timothy D., "Evaluation of Liquid Chromatography–Mass Spectrometry for Routine Proteome Analyses" (2003). Pharmaceutical Sciences Faculty Publications. 491.