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 e… Show more
“…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].…”
Section: Dynamic Ph Junctionmentioning
confidence: 99%
“…As with FASS, Dovichi's group intensively reports ultra-sensitive proteome analysis by PHJ-CZE-nanoESI-MS [63][64][65][66][67].…”
This paper reviews recent 5-years application researches using online sample preconcentration (OSP) methods in capillary electrophoresis (CE) coupled with mass spectrometry (MS). CE-MS has excellent analytical features such as high-resolution separation, rapid analysis time, low sample consumption, and small consumption of organic solvents. Recently, low flow-rate interface for electrospray ionization has been developed and zmol-level sensitivity has been achieved. However, application of CE-MS has still been prevented due to small capacity of sample injection volume. OSP methods have been developed to address this issue and up to 5,000-fold sensitivity improvement was obtained so far. Various application has been carried out such as proteomics, metabolomics, and glycomics, and CE-MS attracts much attention as the highest-performance analytical tool for small-volume bioanalysis. Taking account of further progress in CE-MS, smart and practical application of OSP methods will be more important in various analytical fields.
“…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].…”
Section: Dynamic Ph Junctionmentioning
confidence: 99%
“…As with FASS, Dovichi's group intensively reports ultra-sensitive proteome analysis by PHJ-CZE-nanoESI-MS [63][64][65][66][67].…”
This paper reviews recent 5-years application researches using online sample preconcentration (OSP) methods in capillary electrophoresis (CE) coupled with mass spectrometry (MS). CE-MS has excellent analytical features such as high-resolution separation, rapid analysis time, low sample consumption, and small consumption of organic solvents. Recently, low flow-rate interface for electrospray ionization has been developed and zmol-level sensitivity has been achieved. However, application of CE-MS has still been prevented due to small capacity of sample injection volume. OSP methods have been developed to address this issue and up to 5,000-fold sensitivity improvement was obtained so far. Various application has been carried out such as proteomics, metabolomics, and glycomics, and CE-MS attracts much attention as the highest-performance analytical tool for small-volume bioanalysis. Taking account of further progress in CE-MS, smart and practical application of OSP methods will be more important in various analytical fields.
“…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.…”
Capillary zone electrophoresis (CZE) is emerging as a useful tool in proteomic analysis. Interest arises from dramatic improvements in performance that result from improvements in the background electrolyte used for the separation, the incorporation of advanced sample injection methods, the development of robust and sensitive electrospray interfaces, and the coupling with Orbitrap mass spectrometers with high resolution and sensitivity. The combination of these technologies produces performance that is rapidly approaching the performance of UPLC-based methods for microgram samples and exceeds the performance of UPLC-based methods for mid- to low nanogram samples. These systems now produce over 10,000 peptide IDs in a single 100-minute analysis of the HeLa proteome.
“…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). …”
While capillary zone electrophoresis (CZE) provides dramatically improved numbers of peptide identifications compared with reversed-phase chromatography for bottom-up proteomics of mass limited samples, CZE inevitably produces lower numbers of peptide identifications than RPLC for larger samples. One reason for this poorer performance is the dead time between injection of samples and subsequent appearance of the fastest moving component. This dead time is typically 25% of the separation window in CZE, but is only 5% of the separation window in gradient elution RPLC. This dead time can be eliminated in CZE by use of a multisegment injection mode where a series of samples is analyzed by injecting each sample while the preceding sample is still being separated. In this paper, we demonstrate that capillary zone electrophoresis employing sequential injections can produce a doubling in peptide identification rate with no degradation in separation efficiency.
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