Multiplex proteome quantification method based on isobaric dimethyl labeling

A multiplex proteome quantification method, comprising:

• digesting a protein into a plurality of peptides using a protease that cleaves at a carboxyl side of lysine;
• and
• performing dimethyl labeling of a peptide among the plurality of peptides to produce a plurality of labeled peptides, wherein the dimethyl labeling comprises:
• performing a first dimethyl labeling to a peptide N-terminal under a first acidic condition using a first dimethyl labeling reagent and the second dimethyl labeling to a peptide C-terminal on a Lysine side chain under a first alkaline condition using a second dimethyl labeling reagent to form a first labeled peptide;
• performing a third dimethyl labeling to a peptide N-terminal under a second acidic condition using a third dimethyl labeling reagent and a fourth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a second alkaline condition using a fourth dimethyl labeling reagent to form a second labeled peptide;
• performing a fifth dimethyl labeling to a peptide N-terminal under a third acidic condition using a fifth dimethyl labeling reagent and a sixth dimethyl labeling to a peptide C-terminal on a Lysine side chain under a third alkaline condition using a sixth dimethyl labeling reagent to form a third labeled peptide,
• wherein each of the first dimethyl labeling reagent, the second dimethyl labeling reagent, the third dimethyl labeling reagent, the fourth dimethyl labeling reagent, the fifth dimethyl labeling reagent, and the sixth dimethyl labeling reagent comprises a first compound selected from CH2O, 13CH2O, CD2O, and 13CD2O, and a second compound selected from NaBH3CN and NaBD3CN, and
• the plurality of labeled peptides have a same mass-to-charge ratio;
• ionizing the plurality of labeled peptides and acquiring ionized forms of the plurality of labeled peptides in MS1 of a mass spectrometer;
• separating and fragmenting the ionized forms of the plurality of labeled peptides and acquiring fragment ions in MS2 of the mass spectrometer;
• and
• performing multiplex quantitative analysis based on intensities of the fragment ions in the MS2.