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Accelerating ScienceAccelerating Proteomics / Methods / Quantitation of Peptides in Complex Biological Samples Using Complement Tandem Mass Tag Ions

Quantitation of Peptides in Complex Biological Samples Using Complement Tandem Mass Tag Ions

Written by Megan M. Angelini | Published: 05.27.2013

The challenge of quantitation of peptides via mass spectrometry (MS) has been met in recent years with the advent of isotopic labeling techniques. Quantitative mass spectrometry has a variety of desirable applications, including comparing levels of certain proteins or peptides between clinical samples as indicators of disease states. Previously, it was impossible to compare protein levels between different samples due to the unpredictable nature of the ionization process required for performing MS. By isotopically labeling the proteins of each sample individually and then combining samples to perform MS, however, scientists have been able to compare relative changes in protein amounts between samples. Proteins labeled with isotopic tags have the advantage of being indistinguishable in MS1 data due to their indistinguishable masses. This prevents the spectra from becoming overly complex — a major downfall of metabolic labeling methods. Further fragmentation of these isotopically labeled peptides yields reporter ions that can be used for quantitation. Currently, this type of isotopic labeling has shown the ability to distinguish changes in protein abundance of up to 18 samples.1

Despite the advantages of isotopic labeling for quantitative MS studies, there are also a number of areas that could be improved upon. Using this technique for analysis of multiple complex biological samples is difficult due to the coelution of contaminating ions with the ions of interest. Current methods for combating this problem, which include using an MS3-based approach or using proton-transfer ion-ion reactions, are effective but result in a lowered acquisition rate and decreased sensitivity.

Recently, researchers at Harvard Medical School have found a way to use isotopic labeling for quantitative MS that does not require additional purification steps.2 Instead of using the labeled peptide reporter ions, Martin Wuhr and colleagues use the complement tandem mass tag (TMTc) fragment ion clusters that are formed upon partial loss of the TMT tag on the peptide. These TMTc clusters give the same information on relative peptide abundance as the reporter ions without the contaminant coelution problems. The authors demonstrated that the TMTc technique, using a QExactive Quadropole Orbitrap Mass Spectrometer (Thermo Scientific), was able to accurately quantify labeled yeast peptides in a known yeast/human peptide mixture without coelution interference. This technique lays the groundwork for the possibility of increased multiplexing abilities and parallel quantification in reduced amounts of time using a single MS2 spectrum.


References

1. Dephoure, N., and Gygi, S.P. (2012) ‘Hyperplexing: a method for higher-order multiplexed quantitative proteomics provides a map of the dynamic response to rapamycin in yeast‘, Science Signaling, 5 (217), (p. rs2)

2. Wuhr, M., et al. (2012) ‘Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster‘, Analytical Chemistry, 84 (21), (pp. 9214-9221)

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