Tandem Mass Tag Protein Labeling for Top-Down Identification and Quantification

• Published in: Analytical chemistry (Washington) Vol. 84; no. 1; pp. 161 - 170
• Main Authors: Hung, Chien-Wen, Tholey, Andreas
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 03.01.2012
• Subjects: Analytical chemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Diverse techniques; Electron transfer; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Mass spectrometry; Molecular and cellular biology; Other chromatographic methods; Proteins; Proteins - analysis; Proteomics; Spectrometric and optical methods; Tandem Mass Spectrometry - methods; On line; Ion pair; Electron transfer; Activation; Liquid chromatography; Identification; Protein; Low energy; Characterization; Chemistry; Reversed phase chromatography; Regulation(control); Dynamics; Proteomics; Feasibility; Dissociation; Application; Mass spectrometry; Quantitative analysis; Stationary phase
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202243r

Top-down mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric mass tags in quantitative bottom-up proteomics, we applied the tandem mass tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence tags generated either by low-energy HCD or ETD activation along with the intact protein mass information allow for confident identification of small proteins below 35 kDa. We conclude that the approach presented in this pilot study paves the way for further developments and numerous applications for straightforward, accurate, and multiplexed quantitative analysis in protein chemistry and proteomics.