Evaluation of Liquid Chromatography-Mass Spectrometry for Routine Proteome Analyses

Li Rong Yu, Haleem J. Issaq, Thomas P. Conrads, Takuma Uo, Josip Blonder, George M. Janini, Richard S. Morrison, Timothy D. Veenstra*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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.

Original languageEnglish
Pages (from-to)3331-3342
Number of pages12
JournalJournal of Liquid Chromatography and Related Technologies
Issue number20
StatePublished - 2003
Externally publishedYes


  • Liquid chromatography
  • Mass spectrometry
  • Peptide
  • Proteomites
  • Tandem MS


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