Tandem mass tag protein labeling for top-down identification and quantification

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down mass spectrometry (MS) is powerful for intact protein analysis but rarely combined with quantification.
• Isobaric mass tags (e.g., TMT) are successful in quantitative bottom-up proteomics.

Purpose of the Study:

• To evaluate the feasibility of directly quantifying intact proteins labeled with tandem mass tags (TMT) using top-down proteomics.
• To establish a platform for simultaneous identification and quantification of TMT-labeled intact proteins.

Main Methods:

• Developed a top-down proteomics platform combining ion-pair reversed-phase liquid chromatography with an LTQ-Orbitrap Velos MS.
• Utilized electron-transfer dissociation (ETD) and high-energy collision dissociation (HCD) for protein fragmentation.
• Applied tandem mass tag (TMT) technology for labeling intact proteins.

Main Results:

• TMT-labeled proteins readily dissociated using HCD, releasing reporter ions for quantification.
• Quantification ratios showed a wide dynamic range, independent of protein charge state.
• Protein sequence tags and intact mass information enabled confident identification of proteins <35 kDa.

Conclusions:

• The developed top-down approach enables straightforward, accurate, and multiplexed quantification of intact proteins.
• This method holds significant potential for protein chemistry and proteomics applications.
• Paves the way for further advancements in quantitative protein analysis.