Hydrophilic interaction liquid chromatography (HILIC) in proteomics

Abstract

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. [figure: see text]

Figure

Artistic impression of the HILIC separation mechanism

Fig. 1

Chemical structures of the functional groups in common HILIC stationary phases

Fig. 2

Two-dimensional plots of normalized peptide retention times in HILIC, SCX and RP separation. Both HILIC and SCX have separation mechanisms that are orthogonal to RP, but the clustering of similarly charged peptides in SCX makes this a less optimal first dimension. From the work of Gilar et al. [11] and reproduced with permission from the American Chemical Society

Fig. 3

Distribution of phosphorylated and N-acetylated peptides over ZIC–HILIC fractions. First dimension: ZIC-HILIC, 200 μm × 160 mm, 3.5 μm, 200 Å. Flow rate 1.5 μL/min, 1 min.fractions. Number of peptides: bare line, total; squares, N-acetylated; triangles, phosphorylated. ZIC–HILIC provides clear enrichment of N-acetylated peptides in the initial fractions. From the work of Boersema et al. [12], reproduced with permission from the American Chemical Society

Fig. 4

MALDI-TOF spectra after SPE of a tryptic digest of TIMP-1 by RP (R2, upper spectrum) and HILIC (lower spectrum) microcolumns. HILIC clearly enriches for glycopeptides. This figure was kindly provided by Dr. P. Højrup (similar to [70])

Fig. 5

HILIC separation of GluC-generated long N-terminal peptides from histone H3.2. HILIC primary separation is controlled by the number of acetylations (the three bigger peaks for the +butyrate sample), and within these peaks a secondary separation is observed, relating to the number of methylations (the narrower peaks). Adapted by permission from Macmillan Publishers Ltd: Nature Methods [77]