Abstract: Methods can be conducted to prevent disulfide scrambling in liquid chromatography-mass spectrometry analysis of a protein of interest. In particular, methods can be conducted by employing a NEM analog, such as maleimide, and a heavy isotope counterpart of the NEM analog, such as a heavy isotope substituted maleimide, to alkylate a protein of interest and to alkylate a peptide digest of the protein of interest under conditions that prevent disulfide scrambling and subjecting the samples to a liquid chromatography-mass spectrometry analysis.