Hydrophilic-interaction liquid chromatography (HILIC) is a widely used technique 
for small polar molecule analysis and offers the advantage of improved 
sensitivity in mass spectrometry. Although HILIC is today frequently employed as 
an orthogonal fractionation method for peptides during the proteomic discovery 
phase, it is still seldom considered for quantification. In this study, the 
performances in terms of peak capacity and sensitivity of 3 HILIC columns were 
compared to traditional reversed phase liquid C(18) column in the context of 
targeted quantification of proteotypic peptides using selected reaction 
monitoring mode (SRM). The results showed that the maximum sensitivity in HILIC 
chromatography was achieved by using an amide column without salt buffer and 
that the signal increased compared to classic reversed phase chromatography. 
However, the intensity improvement is quite low compared to the one obtained for 
small molecules. This is due on one hand to a higher matrix effect in HILIC and 
on the other hand to a change of charge states of peptides in organic solvent 
(doubly charged to monocharged). The doubly charged ions can be more readily 
dissociated than singly charged ions, making them ideal for SRM peptide 
quantification. As a result "supercharging" reagents are added to the mobile 
phase to shift from predominant singly charged ions to the more favorable doubly 
charged species. Using such optimized conditions, peptide signal is improved by 
a factor of between two and ten for 88% of the peptides of the 81 peptides 
investigated.