Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics

Andrea F.G. Gargano*, Liana S. Roca, Ryan T. Fellers, Max Bocxe, Elena Domínguez-Vega, Govert W. Somsen

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Recent progress in top-down proteomics has driven the demand for chromatographic methods compatible with mass spectrometry (MS) that can separate intact proteins. Hydrophilic interaction liquid chromatography (HILIC) has recently shown good potential for the characterization of glycoforms of intact proteins. In the present study, we demonstrate that HILIC can separate a wide range of proteins exhibiting orthogonal selectivity with respect to reversed-phase LC (RPLC). However, the application of HILIC to the analysis of low abundance proteins (e.g., in proteomics analysis) is hampered by low volume loadability, hindering down-scaling of the method to column diameters below 2.1 mm. Moreover, HILIC-MS sensitivity is decreased due to ion suppression from the trifluoroacetic acid (TFA) often used as the ion-pair agent to improve the selectivity and efficiency in the analysis of glycoproteins. Here, we introduce a capillary-based HILIC-MS method that overcomes these problems. Our method uses RPLC trap-columns to load and inject the sample, circumventing issues of protein solubility and volume loadability in capillary columns (200 μm ID). The low flow rates and use of a dopant gas in the electrospray interface improve protein-ionization efficiencies and reduce suppression by TFA. Overall, this allows the separation and detection of small protein quantities (down to 5 ng injected on column) as indicated by the analysis of a mixture of model proteins. The potential of the new capillary HILIC-MS is demonstrated by the analysis of a complex cell lysate.

Original languageEnglish
Pages (from-to)6601-6609
Number of pages9
JournalAnalytical Chemistry
Issue number11
Early online date3 May 2018
Publication statusPublished - 5 Jun 2018


This work was financially supported by The Netherlands Organization for Scientific Research by the NWO Veni grant IPA (722.015.009). The authors would like to thank Rob Haselberg (Vrije Universiteit Amsterdam), Peter J. Schoen-makers, and Eva M. Johansson (University of Amsterdam) for their help and valuable discussions and Xiaoli Wang and Linda Lloyd (Agilent Technologies) for the kind gift of the HPLC columns.

FundersFunder number
Netherlands Organization for Scientific Research
Nederlandse Organisatie voor Wetenschappelijk Onderzoek722.015.009


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