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Stable-Isotope Labeling for Protein Quantitation by Mass Spectrometry

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Mass spectrometry has become a routine instrument to identify proteins and peptides from simple or complex samples. Although identification can be confidently determined from a single experiment, quantitation requires multiple replicates and careful analysis. Alternatively, stable isotopes can be used to obtain relative quantitation of proteins and peptides from fewer replicates. Conventionally, half of a sample is labeled with stable isotope and mixed with the other half of unlabeled sample. The mixed sample is analyzed by mass spectrometry and because the stable isotope does not change the chemical properties of the peptide, the intensities of the unlabeled and labeled peptide can be directly compared. Absolute quantitation is obtained by adding a known amount of stable isotope labeled peptide or protein and comparing to an unlabeled counterpart. Stable isotope labeling methodologies can be divided into three categories: Chemical, enzymatic and metabolic. Here we provide an up-to-date review comparing the benefits and drawbacks of all three stable isotope labeling methodologies and briefly describe quantitation software solutions. In addition to quantitation, stable isotopes have also been used to identify post-translational modifications in proteins, identify components of DNA-protein and protein-protein complexes and to distinguish background contaminants from experimental results. Finally, we describe how fragmentation patterns from stable isotope labeled peptide and unlabeled peptides can improve peptide and protein identification and validation.

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Keywords: ICAT; Mass spectrometry; SILAC; chemical; enzymatic; iTRAQ; isobaric; labeling; metabolic; proteomics; quantitation; software; stable isotope

Document Type: Research Article

Publication date: 01 July 2010

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