Analysis of Rates of Multiple Enzymes in Cell Lysates by Electrospray Ionization Mass Spectrometry

Gerber, Scott A.; Scott, C. Ronald; Tureček, František; Gelb, Michael H.

doi:10.1021/ja982878k

Cited by 43 publications

(44 citation statements)

References 7 publications

(6 reference statements)

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“…The ICAT reagents used were synthesized with the following isotopically different substrates: 4,7,10-trioxa-1,13-tridecanediamine (A) (Aldrich, Milwaukee, WI) and 2,2' ,3,3' ,11,11' ,12,12'-octadeutero-4,7,10-trioxa-1,13-tridecanediamine (B) 21 . Synthesis of N- (13-amino-4,7,10-trioxatridecanyl) biotinamide (C) was as follows.…”

Section: Experimental Protocolmentioning

confidence: 99%

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

et al. 1999

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We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

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Section: Experimental Protocolmentioning

confidence: 99%

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

et al. 1999

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“…By means of MS detection, they developed a new method for biomedical applications, in which the activites of two lysosomal enzymes in cell homogenates are simultaneously monitored using specific substrates. 20 Several other multiplexing Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions.…”

Section: Introductionmentioning

confidence: 99%

Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

2012

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“…The mechanism and kinetic response of a whole metabolic pathway could be studied by reproducing conditions that are closer to a complete cellular environment than those provided by common in vitro assays based on isolated enzymes. In a symmetrical approach, a series of substrates containing appropriate molecular tags was synthesized to enable the affinity capture of the respective catalysis products (Gerber et al, 1999(Gerber et al, , 2001. In this way, the individual activities of several enzymes present in whole cell lysates were determined simultaneously for diagnostic purposes.…”

Section: Discussionmentioning

confidence: 99%

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

Fabris

2004

Mass Spectrometry Reviews

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Mass spectrometry (MS) offers many advantages over other established spectroscopic techniques employed for the investigation of processes in condensed phase. The sensitivity, specificity, and speed afforded by MS-based methods enable to obtain very valuable insights into the mechanism of complex dynamic processes. Off-line methods rely on quenching to halt the progress of the reaction of interest and allow for the implementation of a broad range of analytical procedures for sample fractionation, isolation, or desalting. On the contrary, on-line methods are designed to carry out the real-time monitoring of dynamic processes through a continuous uninterrupted analysis of reaction mixtures, with the only caveat that the sample solutions be directly amenable to the available ionization technique. The utilization of rapid mixing devices in direct connection with a mass spectrometer or included in off-line schemes provides access to the initial moments of a reaction, which can offer very important information about the reaction mechanism. This report summarizes the different off- and on-line strategies developed to study chemical and biochemical reactions in solution and obtain kinetic/mechanistic information. The merits of the various experimental designs, the characteristics of the different instrumental setups, and the factors affecting time resolution are discussed with the aid of specific examples, which highlight the contributions of MS to the different facets of the investigation of dynamic processes in condensed phase.

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Analysis of Rates of Multiple Enzymes in Cell Lysates by Electrospray Ionization Mass Spectrometry

Cited by 43 publications

References 7 publications

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

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