Protein sequencing method and reagents

A method of sequencing a plurality of polypeptide molecules in a sample comprising:

• a. affixing the polypeptide molecules in the sample to a plurality of spatially resolved attachment points on a substrate;
• b. contacting the polypeptides with a plurality of probes, wherein each probe comprises an affinity capture reagent that selectively binds to an N-terminal amino acid or a N-terminal amino acid derivative and a fluorescent label, wherein the affinity capture reagent is a variant ClpS polypeptide comprising an amino acid sequence with at least 70% sequence identity to SEQ ID NO:
• 1 or 2;
• c. for a plurality of polypeptides molecule that are spatially resolved and affixed to the substrate, optically identifying the fluorescent label of the probe bound to each polypeptide;
• d. cleaving the N-terminal amino acid or N-terminal amino acid derivative of each of the polypeptides;
• and
• e. repeating steps b) to d) to determine the sequence of at least a portion of one or more of the plurality of polypeptide molecules that are spatially resolved and affixed to the substrate.