Method to Map Protein Landscapes

A method for analyzing two or more polypeptides comprising the steps of:

• a) independently digesting a first sample of a first polypeptide and a first sample of a second polypeptide with a first protease or chemical agent;
• b) independently digesting a second sample of the first polypeptide and a second sample of the second polypeptide with a second protease or chemical agent;
• c) generating tandem mass spectrometry data on each digested polypeptide sample comprising the steps of:
• generating a distribution of precursor ions during MS1 stage ionization, fragmenting precursor ions having a mass-to-charge ratio (m/z) within a selected target m/z range during MS2 fragmentation, thereby generating a plurality of product ions wherein the product ions correspond to amino acid sequences of the polypeptides, and measuring m/z and intensity of the product ions, thereby generating mass spectrometry data for each digested polypeptide sample;
• and
• d) for each polypeptide, combining mass spectrometry data from each digested polypeptide sample of that polypeptide to generate comprehensive mass spectrometry data, wherein the comprehensive mass spectrometry data provides sequence coverage for at least 20% of the full length amino acid sequence for that polypeptide;
• and
• e) generating at least a partial consensus amino acid sequence for each polypeptide from the comprehensive mass spectrometry data or calculating abundances of amino acids for selected portions of each polypeptide from the comprehensive mass spectrometry data.

A system for analyzing a polypeptide having an amino acid sequence comprising:

• a) an ion source for generating ions from a plurality of digested samples of the polypeptide;
• b) ion fragmentation optics in communication with the ion source for generating product ions;
• c) an ion detector in communication with the ion fragmentation optics for detecting ions according to their mass-to-charge ratios;
• d) a mass analyzer in communication with the ion detector, wherein the mass analyzer comprises a software program enabling the mass analyzer to:
• i) measure mass-to-charge ratios and intensity of the detected ions, thereby generating mass spectrometry data for each digested polypeptide sample;
• ii) normalize the measured intensities of the product ions from each digested polypeptide sample;
• and
• iii) combine mass spectrometry data from each digested polypeptide sample to generate comprehensive mass spectrometry data on the polypeptide, wherein the comprehensive mass spectrometry data provides sequence coverage for at least 20% of the full length amino acid sequence of the polypeptide.