Hydrophilic interaction liquid chromatography (HILIC) for the analysis of intact proteins and glycoproteins

Hydrophilic interaction liquid chromatography (HILIC) has always been devoted to the analysis of small polar compounds, glycans and glycopeptides. However, in the last 10 years HILIC has emerged as an analytical tool for the analysis of intact proteins, showing peculiar potentialities in the separation of protein and glycoprotein variants. The great compatibility with mass spectrometry detection and its unique complementarity with reverse phase chromatography makes HILIC an interesting option in intact protein characterization. In this review a comprehensive overview and insight into the use of HILIC for intact protein analysis is given, from retention mechanisms and stationary phases to mobile phase composition, column temperature and injection conditions. After a detailed discussion on method development, the more recent applications of HILIC to intact protein analysis is reviewed, highlighting advantages, limits, and issues of HILIC analysis of intact glycoproteins (such as antibodies and glycoconjugates), histones and lipophilic proteins.