Investigating capillary electrophoresis‐mass spectrometry for the analysis of common post‐translational modifications

Abstract

Capillary electrophoresis coupled to mass spectrometry is a very efficient analytical method for the analysis of post‐translational modifications because of its high separation efficiency and high detection sensitivity. Here we applied CE‐MS using three differently coated separation capillaries for in‐depth analysis of a set of 70 synthetic post‐translationally modified peptides (including phosphorylation, acetylation, methylation, and nitration). We evaluated the results in terms of peptide detection and separation characteristics and found that the use of a neutrally coated capillary resulted in highest overall signal intensity of singly modified peptides. In contrast, the use of a bare‐fused silica capillary was superior in the identification of multi‐phosphorylated peptides (12 out of 15 were identified). Fast separations of approximately 12 min could be achieved using a positively coated capillary, however, at the cost of separation efficiency. A comparison to nanoLC‐MS revealed that multi‐phosphorylated peptides interact with the RP material very poorly so that these peptides were either washed out or elute as very broad peaks from the nano column which results in a reduced peptide identification rate (7 out of 15). Moreover, the methods applied were found to be very well suited for the analysis of the acetylated, nitrated and methylated peptides. All 36 synthetic peptides, which exhibit one of those modifications, could be identified regardless of the method applied. As a final step in this study and as a proof of principle, the phosphoproteome enriched from PC‐12 pheochromocytoma cells was analyzed by CE‐MS resulting in 5686 identified and 4088 quantified phosphopeptides. We compared the characterized analytes to those identified by a nanoLC‐MS proteomics study and found that less than one third of the phosphopeptides were identical, which demonstrates the benefit by combining different approaches quite impressively.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Analysis of post‐translational modifications (PTMs) is still one of the most ambitious tasks in protein analysis with phosphorylation and deamidation being amongst the most challenging PTMs on commonly used RPLC separation systems. The applicability of CZE‐MS for PTM analysis, especially concerning deamidation was demonstrated in recent studies . Faserl et al.…”

“…successfully applied CE‐MS for the analysis of the phosphoproteome of PC‐12 pheochromocytoma cells. They were able to identify 5686 phosphopeptides after digestion and phosphopeptide enrichment by immobilized metal ion affinity chromatography (IMAC) .…”

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015–mid 2018)

“…Additionally, Faserl et al. utilized CE‐MS for the analysis of a set of 70 synthetic post‐transitionally modified peptides (including phosphorylation, acetylation, methylation, and nitration) . Neutrally coated capillary provided highest overall signal intensity of singly modified peptides.…”

“…CE conditions: separation capillary length: 100 cm with porous tip, id: 30 μm, od: 150 μm; BGE: 10% v/v acetic acid; separation voltage: +30 kV. Reprinted with permission from …”

Coupling of CE‐MS for protein and peptide analysis

“…CE conditions: Separation capillary length: 100 cm with porous tip, id: 30 μm; BGE: 10% v/v acetic acid; separation voltage: +30 kV. Reprinted with permission from Copyright 2018 John Wiley and Sons…”