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‡⊥ , and Govert W. Somsen†‡
† Centre for Analytical Science Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
‡ Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
§ Van ’t Hoff Institute for Molecular Sciences, Science Park 904, 1098 XH Amsterdam, Netherlands
∥ Departments of Chemistry and Molecular Bioscience and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208, United States
⊥ Center for Proteomics and Metabolomics, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands
Anal. Chem., Article ASAP
DOI: 10.1021/acs.analchem.8b00382
Publication Date (Web): May 3, 2018
Copyright © 2018 American Chemical Society
*E-mail: [email protected]; Tel:+31-20-5983931; Mailing Address: Dr. Andrea Gargano, Analytical Chemistry, Centre for Analytical Science Amsterdam, VU Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
https://pubs.acs.org/doi/10.1021/acs.analchem.8b00382

Abstract
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.

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