In this work, dimethyl labeling at the protein level was developed to assist the fragmentation of intact proteins using the Q-TOF instrument. It was shown that a! ions were favorably enhanced upon collision-induced dissociation for dimethylated proteins with molecular mass below 20 kDa and without N-terminal modifications. This method is helpful in confirming proteolytic sites located at the N-terminus of proteins. Moreover, this labeling could be incorporated with stable isotopes for comparative profiling at the protein level, in which the heavily labeled and lightly labeled a1 ions were generated from the corresponding proteins upon high-voltage collisions in a broad mass region that covered all of the charge states of the proteins. Using hemoglobin as an example, a linear dynamic range from 1:1 to 1:20 was satisfactorily obtained with an R2 value greater than 0.99. This approach appears to be promising for top-down proteomics.
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