Reversible phosphorylation is one of the most important post-translational modifications of proteins and a key regulator of cellular signaling pathways. Specific enrichment of phosphopeptides from proteolytic digests is a prerequisite for large scale identification of protein phosphorylation by mass spectrometry. Online enrichment of phosphopeptides attracts particular interests due to its automated operation, higher throughput and reproducibility, lower sample loss, and contamination. Here, we report a new type of capillary column developed using surface initiated atom transfer radical polymerization (SI-ATRP) for automated online phosphopeptide enrichment. SI-ATRP modification leads to a surface confined growth of three-dimensional wavelike polymer structure on the inner wall of capillary columns and, therefore, results in largely increased surface area. Furthermore, the noncross-linked flexible polymer chains grown by SI-ATRP create a large internal volume that allows phosphopeptides to penetrate into during enrichment and also facilitate the interaction between the numerous functional groups in the polymer chains and target phosphopeptides. Therefore, highly efficient and specific enrichment is achieved even for a low femtomole of phosphopeptides. The loading capacity is increased more than an order of magnitude compared with that obtained using conventional open tubular capillary columns. The SI-ATRP modified capillary column was successful applied in the online phosphoproteomics analysis of HepG2 cell lysate and resulted in 10 times improved phosphopeptide identification than the previously reported number. Finally, the SI-ATRP technique is compatible with a variety of functional monomers, and therefore, versatile potential applications in reverse phase, ion exchange, and affinity chromatography can be expected.
ASJC Scopus subject areas
- Analytical Chemistry