Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

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Abstract
Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+‐IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of α‐casein with analysis by MALDI‐TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (μRPLC–nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 μg mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with μRPLC‐nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.