Element-Coded Affinity Tags for Peptides and Proteins

Whetstone, Paul A.; Butlin, Nathaniel G.; Corneillie, Todd M.; Meares, Claude F.

doi:10.1021/bc034150l

Cited by 112 publications

(105 citation statements)

References 23 publications

Supporting

Mentioning

105

Contrasting

Order By: Relevance

“…8, the data for the calculation of the detection limit of ICP MS for protein quantification are shown. To this end, different amounts of labeled BSA reaction mixture (16,31,63,125,250, 500, and 1000 ng) were separated using SDS-PAGE. After silver staining, the protein bands were cut out, solubilized, and diluted for FIA/ICP MS analysis.…”

Section: Resultsmentioning

confidence: 99%

“…The synthetic access to DOTA metal complexes has been shown already (16). We used two different labeling reagents named MeCATBnz and MeCAT Bio (Fig.…”

Section: The Reagents and Workflowmentioning

confidence: 99%

“…1). They contain maleimide as the thiolspecific group and DOTA macrocycle, which has been used previously as an immunoglobulin recognition site for affinity purification (16). The two reagents differ in the spacer region, which is short for MeCAT Bnz , whereas MeCAT Bio possesses a biotin-bearing spacer to allow affinity purification (17,18) The reaction of the DOTA macrocycle with lanthanide metal ions (M 3ϩ ) results in stable metal chelate complexes (see Table I) (19,20).…”

Section: The Reagents and Workflowmentioning

confidence: 99%

See 2 more Smart Citations

A Metal-coded Affinity Tag Approach to Quantitative Proteomics

Ahrends

Pieper

Kühn

et al. 2007

Molecular & Cellular Proteomics

154

197

View full text Add to dashboard Cite

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures. Proteomics as a field of research is based on the characterization of an entire proteome of a biological system. A variety of approaches have been developed during the last decades to characterize such mixtures of proteins and peptides, and necessarily, all of them use separation techniques. At the protein level, separation has been achieved using 2-D 1 gel electrophoresis (1) and densitometry of stained proteins or fluorescence detection (2). After digestion of the proteins, peptides were identified using liquid chromatography, mass spectrometry, or both (3, 4). However, this information was only qualitative. It became rapidly evident that quantitative data were definitively required, e.g. for the characterization of dynamic biological systems or the search for biomarkers in clinical proteomics. Subsequently methods have been developed for the quantitative determination of proteins and peptides mainly based on chemical or metabolic isotopic labeling combined with LC/MS n detection (5, 6). Label-free LC/MS quantitative strategies are under development as well (7).Using such techniques, the investigation of changes of the proteome in biological systems has become possible. However, o...

show abstract

Section: Resultsmentioning

confidence: 99%

“…The synthetic access to DOTA metal complexes has been shown already (16). We used two different labeling reagents named MeCATBnz and MeCAT Bio (Fig.…”

Section: The Reagents and Workflowmentioning

confidence: 99%

Section: The Reagents and Workflowmentioning

confidence: 99%

See 1 more Smart Citation

A Metal-coded Affinity Tag Approach to Quantitative Proteomics

Ahrends

Pieper

Kühn

et al. 2007

Molecular & Cellular Proteomics

154

197

View full text Add to dashboard Cite

show abstract

“…AOD has two key elements: 1) an aminooxy group that can form a covalent bond with aldehydes or ketones, and 2) a DOTA (1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid) metal-chelator moiety. AOD is water soluble and can be loaded with a wide choice of monoisotopic rare earths to generate a series of mass tags with large mass defects 16 and nearly identical chromatographic behavior 17 . Confirmation of the labeling is achieved using Western blotting.…”

Section: Introductionmentioning

confidence: 99%

Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

et al. 2006

Self Cite

View full text Add to dashboard Cite

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products.After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were 3 found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend this methodology to study disease-related oxidation systems.

show abstract

“…Other types of fusion tags are also available including maltose-binding protein (di Guan et al, 1988), thioredoxin (LaVallie et al, 1993), NusA (Whetstone et al, 2004), GB1 domain for protein G (Davis et al, 1999), and others (Balbas, 2001;Thorn et al, 2000). The decision to use any of these tagging methods depends mainly on the needs of of the researcher.…”

Section: Recombinant Tagged Proteinsmentioning

confidence: 99%