Selective sampling of multiply phosphorylated peptides by capillary electrophoresis for electrospray ionization mass spectrometry analysis

Abstract

The ionization of phosphorylated peptides in positive ion mode mass spectrometry is generally less efficient compared with the ionization of their non-phosphorylated counterparts. This can make phosphopeptides much more difficult to detect. One way to enhance the detection of phosphorylated proteins and peptides is by selectively isolating these species. Current approaches of phosphopeptide isolation are based on the favorable interactions of phosphate groups with immobilized metals. While these methods can be effective in the extraction, they can lead to incomplete sample recovery, particularly for the most strongly bound multiply phosphorylated components. A non-sorptive method of phosphopeptide isolation using capillary electrophoresis (CE) was recently reported [Zhang et al., Anal. Chem. 77 (2005) 6078]. The relatively low isoelectric points of phosphopeptides cause them to remain anionic at acidic sample pH. Hence, they can be selectively injected into the capillary by an applied field after the electroosmotic flow (EOF) is suppressed. The technique was previously coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, the exploitation of selective sampling in conjugation with electrospray ionization mass spectrometry (ESI-MS) is presented. The transition was not immediately straightforward. A number of major alterations were necessary for ESI interfacing. These adaptations include the choice of a suitable capillary coating for EOF control and the incorporation of organic solvent for efficient ESI. As expected, selective injection of phosphopeptides greatly enhanced the sensitivity of their detection in ESI-MS, particularly for the multiply phosphorylated species that were traditionally most problematic. Furthermore, an electrophoretic separation subsequent to the selective injection of the phosphopeptides was performed prior to analysis by ESI-MS. This allowed us to resolve the multiply phosphorylated peptides present in the samples, predominantly based on the number of phosphorylation sites on the peptides.