Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry for Top-Down Characterization of the <i>Mycobacterium marinum</i> Secretome

Zhao, Yimeng; Sun, Liangliang; Champion, Matthew M.; Knierman, Michael D.; Dovic̀hi, Norman J.

doi:10.1021/ac500092q

Cited by 50 publications

(59 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the offline strategy, proteins are first separated either by gel-based methods, liquid Capillary electrophoresis (CE) can be directly interfaced with mass spectrometers via ESI and used for intact protein characterization taking advantage of an excellent resolution size-sorted capacity and a good sensitivity (105).Top down approaches using CE allow elucidation of complex post-translational modifications to generate information on protein isoforms (59). However, top down proteomics allow limited proteome coverage identification compared with a bottom up approach.…”

Section: Top Down Proteomicsmentioning

confidence: 99%

Redox Proteomics Applied to the Thiol Secretome

Ghezzi

Chan

2017

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Word count: 6800 excluding references Number of references: 113Number of greyscale and color illustrations: 7 greyscale and 5 color (online only)Ghezzi 2 AbstractStudies of redox proteomics to identify glutathionylated proteins have so far been focused on intracellular proteins. However, plasma proteins have been described as glutathionylated. The present review discusses the redox state of protein cysteines in relation to their cellular distribution. We focus in particular on the redox state of proteins secreted under inflammatory conditions and that may act as soluble mediators (cytokines). We describe the various approaches used to detect secreted glutathionylated proteins, the only thiol modification studied so far in secreted proteins, and the specific problems presented in the study of the secretome. This review will deal with a particular aspect of redox proteomics, the analysis of secreted proteins and their possible function. Ghezzi 3 Redox state of proteins in the context of their localization Redox proteomics of the thiol proteomeAccording to the Oxford English Dictionary, proteomics is "the study or analysis of the set of proteins expressed by an organism" and we commonly apply the term to high-throughput techniques of protein identification. As such, the history of redox proteomics started in 2002 when various groups published the first reports of the identification of oxidized proteins by modifying the existing proteomic techniques.Two of these papers were aimed at carbonylated proteins (19,20) and others at protein glutathionylation (33,37,61) or overall protein thiol oxidation (60) .This review will deal with the redox states of protein thiols. Biochemistry textbooks and protein databases classify cysteines in proteins as either free or engaged in a disulfide bond. However, "free" cysteines can be reversibly or irreversibly oxidized to various forms. In particular, in addition to forming structural disulfides, cysteines can form transient, reversible disulfides with another protein cysteine, with free cysteine (cysteinylation) or with the cysteine in the tripeptide glutathione (GSH;; glutamylcystenyl-glycine). Fig. 1 lists the most important oxidation state of sulfur in cysteine.These forms of oxidation, due to their reversibility, are of great interest in the context of redox regulation, as a means by which the redox state of the cell can regulate protein functions (43-45).Specific proteins undergoing glutathionylation were described even before the term "proteomics" became popular (in the late 1990s, according to Google books ngram viewer -https://books.google.com/ngrams). Two proteins have been identified as undergoing glutathionylation in two separate papers, an unidentified 30 kDa cytosolic Ghezzi 4protein in oxidant-treated rat liver (79), and actin in human neutrophils during oxidative burst (22). Although these were not the result of high-throughput studies, some of the technologies used were then adapted for two-dimensional gels for further studies and are at the basis of current red...

show abstract

Section: Top Down Proteomicsmentioning

confidence: 99%

Redox Proteomics Applied to the Thiol Secretome

Ghezzi

Chan

2017

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

show abstract

“…[6] Later, Zhao et al further applied the CZE-MS/MS system for top-down proteomics of Mycobacterium marinum secretome and yeast proteome. [7, 8] Coupling offline RPLC fractionation to CZE-MS/MS identified 580 proteoforms from a yeast lysate. In total, 23 RPLC fractions were analyzed by CZE-MS/MS and up to 180 proteoforms could be identified with single-shot CZE-MS/MS.…”

Section: Introductionmentioning

confidence: 99%

Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms

et al. 2017

Self Cite

View full text Add to dashboard Cite

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS approached 1-μL loading capacity, 90-min separation window and high peak capacity (~280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal and some post-translational modifications including oxidation and acetylation were detected.

show abstract

“…Taken with permission from Ref. [23] two years, a few studies have demonstrated that efficient CE separations can be combined with a top-down proteomics approach [62][63][64]. In all cases, coupling was achieved using the interface designed by Dovichi.…”

Section: Top-down Proteomicsmentioning

confidence: 99%

“…High concentrations of standard proteins (µM-range) were injected to demonstrate the feasibility of top-down proteomics in CE-MS. Moreover, this approach has also been applied to characterize unknown proteins in both cell lysates [62] and culture filtrates [63]. No indications about minimum protein amounts or concentrations required for good sequence coverage is given.…”

Section: Top-down Proteomicsmentioning

confidence: 99%