Fully Unattended Online Protein Digestion and LC–MS Peptide Mapping

LC–MS based peptide mapping, i.e., proteolytic digestion followed by LC–MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC–MS peptide mapping. The method generates LC–MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC–MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC–MS analysis.