Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

Roland S Annan,Dean E Mcnulty

doi:10.1074/mcp.m700543-mcp200

Roland S Annan, Dean E Mcnulty

Open Access

https://doi.org/10.1074/mcp.m700543-mcp200

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Abstract

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

Highlights

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components
Gilar et al [19] have shown that hydrophilic interaction chromatography (HILIC) has the highest degree of orthogonality to RPLC of all commonly used peptide separation modes. This and the suitability of HILIC for the highly efficient separation of polar molecules prompted us to investigate the potential of HILIC as part of a multidimensional separation strategy for phosphoproteomics. In this current study we demonstrate that HILIC as a first dimension separation for 2D LC proteomics provided an equivalent number of peptide and protein identifications to strong cation exchange (SCX) for cell lysates
When we analyzed peptides from an IMAC enriched tryptic digest of a HeLa cell lysate we found that the majority of the UV absorbance eluted in the same region as the casein phosphopeptides (Fig. 3B)

Summary

HILIC Reduces the Complexity of the Phosphoproteome

Under typical SCX conditions, most tryptic phosphopeptides will elute in the 1ϩ fractions, whereas the bulk of non-phosphorylated tryptic peptides will elute in the 2ϩ and higher fractions. Gilar et al [19] have shown that HILIC has the highest degree of orthogonality to RPLC of all commonly used peptide separation modes This and the suitability of HILIC for the highly efficient separation of polar molecules prompted us to investigate the potential of HILIC as part of a multidimensional separation strategy for phosphoproteomics. In this current study we demonstrate that HILIC as a first dimension separation for 2D LC proteomics provided an equivalent number of peptide and protein identifications to SCX for cell lysates. This ensures fair competition between the phosphopeptides and the non-phosphopeptides in each fraction and provides highly enriched phosphopeptide pools without the need for derivatization or additives

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Unique sites