Compositions and methods for analysis of protein sequences and post-translational modifications

A method for characterizing a protein, said method comprising:

• optionally denaturing the protein;
• dissolving said protein in a digestion buffer;
• passing the digestion buffer comprising said protein through a reaction chamber comprising at least one hydrolyzing agent, wherein said protein contacts said hydrolyzing agent and is present in the chamber for a period of time (t) sufficient to produce protein fragments and digestion of said protein occurs in the chamber, wherein the passing of the digestion buffer comprising the protein through the chamber is done at a flow rate which determines the digestion of the protein in the reaction chamber;
• and
• performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein, wherein said characterization is selected from sequencing, identifying post-translational modifications (PTMs), and locating disulfide bonds,
• wherein said digested protein is subjected to electron transfer dissociation (ETD)/ion-ion proton transfer (IIPT) to identify the disulfide-containing fragments and N- and C-terminal sequences and localize the disulfide(s) within/connecting different domains.