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

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-g equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome. Molecular & Cellular Proteomics 7:971-980, 2008.

Analytical strategies for phosphoproteomics

“…Kokubu and co-workers showed that 0.1% TFA, 50% ACN as IMAC loading buffer reduced the level of nonspecific binding and improved the specificity of the method toward phosphopeptides [56]. Yet another approach to improve the performance of IMAC is to reduce the complexity of the peptide samples by prefractionation using methods such as IEF [57][58][59][60], Ion Exchange Chromatography [41,43,46,61] or hydrophilic interaction chromatography (HILIC) [62] prior to IMAC purification.…”

“…These hydrogen bonds can subsequently be disrupted by decreasing the organic environment in the mobile phase and peptides will elute according to their polarities (hydrophilicities). McNulty and Annan introduced HILIC as a peptide prefractionation technique prior to phosphopeptide enrichment by IMAC [62]. Using a TSKgel Amide-80 HILIC column and a shallow inverse organic gradient of ACN, water and TFA, they fractionated 1 mg of tryptic peptides from HeLa cells, which had been starved and treated with the phosphatase inhibitor, calyculin A.…”

“…Using a TSKgel Amide-80 HILIC column and a shallow inverse organic gradient of ACN, water and TFA, they fractionated 1 mg of tryptic peptides from HeLa cells, which had been starved and treated with the phosphatase inhibitor, calyculin A. They tested the efficiency of HILIC as a phosphopeptide enrichment technique and compared it to SCX and IMAC as well as to the combination of HILIC with IMAC for more efficient phosphopeptide recovery [62]. They found that peptides were uniformly distributed throughout the HILIC gradient, containing mainly short, hydrophobic peptides in the early fractions and larger, hydrophilic, and highly acidic peptides in the later fractions.…”

“…They found that the combination of HILIC and IMAC improved phosphopeptide recovery significantly. When IMAC was performed prior to HILIC, a high level of nonspecific binding due to nonphosphorylated peptides was observed, whereas HILIC separation prior to IMAC enrichment improved the phosphopeptide selectivity to more than 99% [62]. Thus, HILIC seems highly efficient for prefractionation of highly complex samples prior to specific phosphopeptide enrichment methods such as IMAC, TiO 2 chromatography, or SIMAC.…”

What does the future hold for top down mass spectrometry?

“…An incredible number of HILIC stationary phases have been reported for diverse selective and specialized applications to a range of analytes. Accordingly, in recent years, HILIC has experienced a dramatic growth for proteomic applications, ranging from the separation of complex peptide mixtures [28,29], to the enrichment of subproteomes, such as phosphorylated [30] or glycosylated peptides [31], or N-terminally acetylated peptides [29], usually before Bottom Up mass spectrometry is applied.…”