Detachable Strong Cation Exchange Monolith, Integrated with Capillary Zone Electrophoresis and Coupled with pH Gradient Elution, Produces Improved Sensitivity and Numbers of Peptide Identifications during Bottom-up Analysis of Complex Proteomes

Abstract

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with on-line pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction, and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3,381 peptides were identified from 50 ng of the digest in a 2.5 hour analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the on-line pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

Recent Studies on Online Sample Preconcentration Methods inCapillary Electrophoresis Coupled with Mass Spectrometry

“…According to the preference of PHJ, proteins, peptides, and amino acids are mostly analyzed which have moderate pKa or pI values [63][64][65][66][67][68][69][70][71][72][73]. As with FASS, Dovichi's group intensively reports ultra-sensitive proteome analysis by PHJ-CZE-nanoESI-MS [63][64][65][66][67].…”

“…As with FASS, Dovichi's group intensively reports ultra-sensitive proteome analysis by PHJ-CZE-nanoESI-MS [63][64][65][66][67].…”

Capillary zone electrophoresis for bottom‐up analysis of complex proteomes

“…Elution buffers of increasing pH can be coupled with the on-line strong cation exchange monolithic preconcentrator to generate fractions for CZE analysis [38]. There is remarkably little overlap in peptide identifications between successive pH bumps, and the system provides a powerful tool to analyze complex proteomes.…”

Multisegment injections improve peptide identification rates in capillary zone electrophoresis-based bottom-up proteomics

“…As a result of these studies, it is clear that CZE offers several advantages compared to RPLC for this analysis: CZE produces many more peptide and protein identifications from mass limited samples (17), a simple model has been developed for the migration time of peptides to increase confidence of identifications (19), and on-column digestion has been developed to simplify sample manipulations (20–21). …”