Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments

He, Yide; Esteban-Fernández, Diego; Neumann, Boris; Bergmann, Ulf; Bierkandt, Frank S.; Linscheid, Michael

doi:10.1016/j.jprot.2015.12.010

Cited by 8 publications

(6 citation statements)

References 28 publications

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“…Western blot was used as well in combination with immunoassay for the analysis of labeled cytochromes, in high throughput microarrays, and for the quantification of plant thylakoid proteins by means of Mal‐DOTA labeled antibodies . Quantification directly from gels was used often from the very first to the recent studies of the impact of heat shock experiments of the protein abundance in E coli …”

Section: Imaging Mass Spectrometrymentioning

confidence: 99%

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Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

Linscheid

2018

Mass Spectrometry Reviews

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To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive P with the "cold" natural P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years.

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Section: Imaging Mass Spectrometrymentioning

confidence: 99%

“…132 Quantification directly from gels was used often from the very first 51 to the recent studies 73 of the impact of heat shock experiments of the protein abundance in E coli. 133…”

Section: Maldi Msimentioning

confidence: 99%

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

Linscheid

2018

Mass Spectrometry Reviews

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“…The exposed free thiol groups can subsequently be conjugated to a linker molecule (e.g. maleimide) which is connected to the metal label 15 . Unfortunately, the reduction reactions are not entirely selective and may generate a mixture of antibody fragments and conjugates lacking affinity or metal label.…”

Section: Introductionmentioning

confidence: 99%

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays

et al. 2019

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This study presents a novel method based on size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) for the characterisation and quantification of immunoassays employing lanthanide-labelled antibodies. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD=3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid high-resolution chromatographic method with separation times under 7 minutes of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided new insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153 Eu labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156 Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated only 1.9% from the immunochemical approach employing 153 Eu labelled polyclonal antibodies.

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“…MeCAT‐Click was developed as a toolbox for labeling of cysteine‐containing proteins, because the two‐step process—first cysteines are derivatized with an alkyne linker, and then the lanthanide loaded DOTA‐azide is attached by click chemistry—enhances the labeling efficiency in complex samples (Fig. S1 and Schemes S1 and S2 in the Supporting Information) and has been successfully applied to screen the differential expressed proteins in thermally stressed Escherichia coli …”

Section: Introductionmentioning

confidence: 99%

“…S1 and Schemes S1 and S2 in the Supporting Information) and has been successfully applied to screen the differential expressed proteins in thermally stressed Escherichia coli. [13] The study of fragmentation behavior of the labeled species is crucial for identification of specific characteristic product ions, termed 'reporter ions' and to locate and quantify labeled species.…”

Section: Introductionmentioning

confidence: 99%

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA‐based labels and N‐termini DOTA‐labeled peptides

El‐Khatib

Esteban-Fernández

et al. 2017

J. Mass Spectrom.

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1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln-MeCAT-Click and Ln-DOTA-Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post-translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln-MeCAT-Click and Ln-DOTA-Dimedone was studied using collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher-energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD > HCD (normal energy level) > CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE > 250%) resulted in a significant increase in reporter ion production (33-54%). This new strategy was successfully applied to generate label-specific reporter ions for DOTA amino labeling at the N-termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd.

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Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments

Cited by 8 publications

References 28 publications

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA‐based labels and N‐termini DOTA‐labeled peptides

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