Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum...

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Bibliographic Details
Published in:Analytical chemistry (Washington) Vol. 86; no. 8; pp. 3817 - 3824
Main Authors: Horie, Kanta, Kamakura, Takeo, Ikegami, Tohru, Wakabayashi, Masaki, Kato, Takashi, Tanaka, Nobuo, Ishihama, Yasushi
Format: Journal Article
Language:English
Published: United States American Chemical Society 15-04-2014
Subjects:
Analytical chemistry
Capillarity
Chromatography
Chromatography, Liquid - instrumentation
Chromatography, Liquid - methods
HeLa Cells
Humans
Kinetics
Liquid chromatography
Mass Spectrometry - methods
Neoplasm Proteins - chemistry
Nucleosides
Peptides
Peptides - chemistry
Permeability
Proteins
Proteomics
Proteomics - methods
Silica
Silicon Dioxide
Trypsin - chemistry
Urea - chemistry
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Summary:A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10–13 m2, which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.
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ISSN:0003-2700
1520-6882
DOI:10.1021/ac4038625