Multiplexed cysteine-reactive mass tags reduce sample complexity for better sensitivity and dynamic range.

Thermo Scientific cysTMT* Mass Tag Reagents are thiol-reactive isobaric tags for labeling reduced cysteine residues in order to perform multiplex quantitative mass spectrometry (MS) analysis of protein expression. When used in combination with the Immobilized Anti-TMT Antibody Resin, the cysTMT tags allow specific labeling, enrichment and quantitation of cysteine-containing peptides and proteins from complex mixtures of protein samples.

Highlights of CysTMT Mass Tag Reagents:

• Specific – only reacts with reduced sulfhydryl groups
• Monitorable – reaction progress and labeling efficiency can be monitored by UV absorption
• Flexible – options for duplex isotopic (MS) or sixplex isobaric (MS/MS) quantitation
• Sensitive – improved dynamic range by targeted enrichment of labeled peptides

About Cysteine-Reactive Mass Tag Reagents

Thermo Scientific TMT Reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry. Isobaric TMT Reagents share identical structures, including a peptide-reactive labeling group, a spacer arm and an MS/MS quantitative reporter. During MS/MS analysis of peptides, each isobaric tag produces a unique reporter ion that makes relative quantitation possible.

The approach of thiol-targeted labeling, affinity enrichment and quantitation is similar to isotope-coded affinity tags (ICAT). However, cysTMT Reagents offer several advantages over ICAT including better specificity, greater multiplexing options and a unique capture mechanism for easier recovery of labeled peptides. In addition, pyridyldithiol-activated cysTMT reagents are not light-sensitive, unlike iodoacetamide labeling reagents, and they react specifically with reduced thiols. Upon labeling, pyridine-2-thione is released and can be detected by UV absorbance (343nm) in order to monitor reaction progress and efficiency. The cysTMT Reagents are compatible with samples containing urea or detergents and allow labeling and mixing of intact protein samples before or after digestion.

Because of the low abundance of free cysteine residues in protein mixtures, it is beneficial to enrich samples before MS analysis. For TMT-labeled proteins and peptides, this can be accomplished using an immobilized anti-TMT monoclonal antibody which has high affinity (Kd >100pM) for the reporter region of the TMT Reagent. Enrichment of labeled peptides by this method results in fewer peptides identified per protein but can significantly increase the number of unique proteins identified, making identification and quantitation of low abundance proteins easier. In addition to improving the dynamic range of sample analysis, cysTMT reagents can also be used to assess the oxidation or S-nitrosylation state of cysteine residues.

Specific cysteine residue labeling with Thermo Scientific cysTMT Mass Tag Reagents. The thiol-reactive cysTMT mass tags specifically react with reduced thiol moieties. During the reaction, pyridine-2-thione is generated allowing progress of the reaction to be monitored by UV absorbance (343nm).

Structure and functional regions of the Thermo Scientific CysTMT Reagents. Panel A: Structure of the cysTMTzero Mass Tag Reagent showing the mass reporter, mass normalizer and cysteine-reactive regions common to all of the cysTMT mass tags. Panel B: Structures for each of the six isobaric cysTMT mass tag reagents with positions of 13C and 15N heavy isotopes identified (*). Sixplex quantitation may be performed when samples are labeled with the cysTMTsixplex 126-131 reagents. Duplex isotopic MS quantitation is performed on peptides labeled with cysTMTzero (Panel A.) or cysTMTsixplex-127. Also shown are the sites for MS/MS fragmentation that releases a reporter ion upon collisional-induced dissociation (CID) or electron-transfer dissociation (ETD).

Workflow for multiplex quantification of protein expression using Thermo Scientific cysTMT Reagents and LC-MS/MS. Reduced proteins from up to 6 samples are labeled with different cysTMT Mass Tags. After mixing the samples together, unincorporated reagent is removed by gel electrophoresis, gel filtration, strong-cation exchange chromatography or acetone precipitation. Following digestion, cysTMT-labeled peptides are enriched before LC-MS/MS.

Specific enrichment of cysTMT-labeled peptides. A synthetic cysteine-containing peptide [GLP-1 (7-17)-Cys, AnaSpec, Inc.] (1mg/mL) was reduced with immobilized TCEP for 45 minutes at room temperature and labeled for 2 hours with 0.85 molar equivalents of Thermo Scientific cysTMTzero Reagent. The cysTMT-labeled peptide (1nmol) was spiked into a BSA tryptic digest (100µg) and the sample was processed with the Thermo Scientific TMT Enrichment Kit (Part No. 90077) before LC-MS/MS analysis. Panel A. MS chromatogram of the BSA tryptic digest containing the cysTMT-labeled GLP peptide before enrichment. Panel B. MS chromatogram of the sample after enrichment with the TMT Enrichment Kit showing a single peak corresponding to the cysTMT-labeled GLP peptide.

Thermo Scientific cysTMT Reagents enable detection of more unique proteins from complex samples. To evaluate enrichment in a complex sample, HeLa cell lysate (100µg) was labeled with the cysTMTzero Reagent, desalted and digested. Samples were then analyzed by MS without enrichment (Total) or processed using the TMT enrichment kit before MS analysis (Enriched). Panel A: The total number of peptides identified in each sample group showing the number of peptides unique and common to each group (Total or Enriched). Panel B: The total number of proteins identified in each sample group showing the number of proteins unique and common to each group. Conclusion: Enrichment of labeled peptides resulted in fewer total peptides identified per protein (1.9 versus 5.2 peptides per protein), but nearly twice as many unique proteins were identified because the reduction in peptide sample complexity allows identification of low abundance proteins.

References:

1. Thompson, A., et al. (2003). Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75:1895-204.
2. Dayon, L., et al. (2008). Relative quantification of protein in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal. Chem. 80(8):2921-31.
3. Gygi, S.P., et al. (1999). Quantitative analysis of complex protein mixtures using isotopecoded affinity tags. Nat. Biotech. 17:994-9.
4. Sethuraman, M., et al. (2004). Isotope-coded affinity tag approach to identify and quantify oxidant-sensitive protein thiols. Mol. Cell. Proteomics 3:273-8.
5. Meza, J.E., et al. (2003). Essential cysteine-alkylation strategies to monitor structurally altered estrogen receptor as found in oxidant-stressed breast cancers. Anal. Biochem. 20(1):21-31.
6. Chen, Y-Y., et al. (2008). Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation. J. Biol. Chem. 283:35265-72.

Related Resources:

Mass Spec Sample Preparation Handbook
Mass Spectrometry Sample Prep Workflow

Related Products:

Immobilized TCEP and other reducing agents
Zeba Spin Desalting Columns
Halt Protease and Phosphatase Inhibitors
TMT amine-Reactive Tandem Mass Tag Reagents
SILAC Isotope Labeling Reagents and Kits

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