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

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.