Abstract:We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) CE-MS/MS procedure which employs a high sensitivity porous ESI sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency we primarily… Show more
“…Similar complementary peptide identification was found in recent CE and nano-LC studies by our group and other researchers (32,39,58). CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass.…”
Section: Discussionsupporting
confidence: 87%
“…CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass. Additionally, the Yates Lab attributed gains in sensitivity to lower noise levels with CE, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides relative to LC (58). These trends may be also explained by the difference in electrospray conditions between the methods.…”
We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N ␣ -terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example. Molecular & Cellular
“…Similar complementary peptide identification was found in recent CE and nano-LC studies by our group and other researchers (32,39,58). CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass.…”
Section: Discussionsupporting
confidence: 87%
“…CE shows a clear bias toward basic, hydrophilic peptides of low molecular mass. Additionally, the Yates Lab attributed gains in sensitivity to lower noise levels with CE, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides relative to LC (58). These trends may be also explained by the difference in electrospray conditions between the methods.…”
We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N ␣ -terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example. Molecular & Cellular
“…However, since only the porous end of the separation capillary is housed in the reservoir, the passage of ions from the reservoir through the pores in the separation capillary is achieved without dilution of the BGE. A number of groups have utilized this interface in proteomics experiments [1, [66][67][68][69][70]. The interface is sold commercially via SCIEX (Toronto, Canada) and marketed for CZE-MS applications as a part of SCIEX's CESI platform.…”
“…As with FASS and PHJ, major application field of tITP-CE-MS includes proteomics [82][83][84][85][86][87][88]. Wang et al reported combination of reverse-phase solid phase microextraction with tITP-CZE-nanoESI-MS [82].…”
mentioning
confidence: 99%
“…Wang et al reported combination of reverse-phase solid phase microextraction with tITP-CZE-nanoESI-MS [82]. They succeeded in identification of up to 370 proteins from 50 ng of Xenopus laevis proteins.…”
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.
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