A multidimensional chromatography technology for in-depth phosphoproteome analysis

Abstract

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

Fig. 1.

The use of HILIC for phosphopeptide separation. A, schematics of a multidimensional chromatography technology used to purify, separate, and analyze the phosphopeptides purified from proteolyzed cell lysate. B, the gradients used in HILIC (dashed lines) and the UV absorbance of the peptides in the bound fraction (dark blue line) and flow-through (light blue line) of IMAC separated by the HILIC. There is a partial overlap between the unphosphorylated and phosphorylated peptides. The HILIC fractions from 13–32 containing mostly phosphopeptides, colored in red, were analyzed by RP-HPLC-MS/MS. The peak appearing before 10 min is due to injection and contains few peptides. Unphosphorylated peptides were mostly found in the fractions indicated by asterisks.

Fig. 2.

Comparison of the RP-HPLC profile and phosphopeptide identification from three adjacent HILIC fractions. A, ion abundances of the peptides detected by RP-HPLC-MS for HILIC fractions 14–16. Numerous peptide ions appear throughout RP-HPLC in each fraction that are different from one another. B, Venn diagram indicates the overlaps of the identified phosphopeptides in HILIC fractions 14–16. Although there is an ∼20% overlap between adjacent fractions, few overlaps (∼5%) were found between fractions 14 and 16.

Fig. 3.

Summary of the phosphopeptides and phosphoproteins identified from yeast. A, phosphopeptides were purified using 6 mg of total proteins as a starting material. After separation using HILIC, 20 fractions were analyzed by RP-HPLC-MS/MS and searched using SEQUEST and InsPecT. This led to the identification of 8764 unique phosphopeptides from 2278 proteins. Possible redundancy due to charge state, oxidative state, or even possible ambiguity on phosphorylation site assignment was removed to calculate the number of unique phosphopeptides reported here. The false discovery rate is less than 1% as judged by the target-decoy strategy. B, Venn diagram of the phosphopeptides identified using InsPecT and SEQUEST, indicating that these two search tools are partially complementary to each other. C, Venn diagram of the number of phosphopeptides of Rad9 identified using the HILIC, SCX, and pulldown approaches. D, Venn diagram of the number of phosphopeptides of Mrc1 identified using the HILIC, SCX, and pulldown approaches. The amount of starting material used in each case is indicated. In this case, manual examination of each phosphopeptide of Rad9 and Mrc1 was performed.

Fig. 4.

Analysis of the SQ/TQ phosphorylation in the budding yeast. A, comparison of the number of SQ/TQ phosphorylated peptides using either the SCX-based or the HILIC-based technology. About 4-fold more SQ/TQ phosphorylated peptides were found here. B, a summary of the SQ/TQ phosphorylated proteins with known roles in DNA replication, repair, and DNA damage checkpoint. Those known Mec1/Tel1 targets are colored in blue, whereas the newly identified potential targets are colored in green.