Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.