![Sample our Physical Sciences journals, sign in here to start your FREE access for 14 days]({"type":"image" , "src" : "/sda/110819/TOC-Subject-Sample-2021-Physical-Sciences.jpg"})
Abstract
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.
Acknowledgments
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1‐CO‐12400. Dr. Morrison wishes to thank NIH (Grant No. NS35533) for funding of this work.
By acceptance of this article, the publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty‐free license and to any copyright covering the article. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.