Development of microwave-assisted acid hydrolysis of proteins using a commercial microwave reactor and its combination with LC–MS for protein full-sequence analysis

Abstract

Microwave-assisted acid hydrolysis (MAAH) can be used to degrade a protein non-specifically into many peptides with overlapping sequences which can be identified by mass spectrometry (MS) to produce a sequence map that covers the full sequence of a protein. The success of this method for protein sequence analysis depends on the proper control of the MAAH process, which is currently done using a household microwave oven. However, to meet the regulatory or good laboratory practice (GLP) requirement in a clinical or pharmaceutical laboratory, using a commercial microwave device is often required. In this paper, we report a method of performing MAAH using a CEM Discover single-mode microwave reactor. It is shown that, using an optimized protocol for MAAH, reproducible results comparable to those obtained using a household microwave oven can be generated using the commercial reactor. To illustrate the potential applications of MAAH MS for characterizing clinically relevant proteins, this method was applied, for the first time, to map the amino acid sequences of normal and sickle-cell human hemoglobin as well as bovine hemoglobin. Full sequence coverage was readily achieved from 294 and 266 unique peptides matched to the alpha and beta subunits of normal hemoglobin, respectively, 334 and 265 unique peptides matched to the alpha and beta submit units of sickle-cell hemoglobin, and 377 and 224 unique peptides matched to the alpha and beta subunits of bovine hemoglobin. This method opens the possibility for any laboratory to use a commercial laboratory equipment to perform MAAH MS for protein full-sequence analysis.