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Int J Mass Spectrom. 2015 Nov 30;391:105-114.

Evaluating the use of HILIC in large-scale, multi dimensional proteomics: Horses for courses?

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Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.


Despite many recent advances in instrumentation, the sheer complexity of biological samples remains a major challenge in large-scale proteomics experiments, reflecting both the large number of protein isoforms and the wide dynamic range of their expression levels. However, while the dynamic range of expression levels for different components of the proteome is estimated to be ∼107-8, the equivalent dynamic range of LC-MS is currently limited to ∼106. Sample pre-fractionation has therefore become routinely used in large-scale proteomics to reduce sample complexity during MS analysis and thus alleviate the problem of ion suppression and undersampling. There is currently a wide range of chromatographic techniques that can be applied as a first dimension separation. Here, we systematically evaluated the use of hydrophilic interaction liquid chromatography (HILIC), in comparison with hSAX, as a first dimension for peptide fractionation in a bottom-up proteomics workflow. The data indicate that in addition to its role as a useful pre-enrichment method for PTM analysis, HILIC can provide a robust, orthogonal and high-resolution method for increasing the depth of proteome coverage in large-scale proteomics experiments. The data also indicate that the choice of using either HILIC, hSAX, or other methods, is best made taking into account the specific types of biological analyses being performed.


2D-LC; Chromatography; HILIC; HILIC, hydrophilic interaction liquid chromatography; Mass-spectrometry; Multi-dimensional proteomics; PTM; PTM, post-translational modification; RP, reverse-phase; hSAX, hydrophilic strong anion exchange

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