Thermal denaturation: a useful technique in peptide mass mapping

The use of thermal denaturation of proteins prior to in-solution digestion and mass spectral peptide mass mapping is reported. Thermal denaturation is preferred over chemical denaturation because it does not require purification/concentration prior to mass spectral analysis. Enzymatic digestions of proteins that are resistant to proteolysis are significantly enhanced by thermal denaturation. Native proteins that are sensitive to proteolysis show similar or slightly lower digestion yields following thermal denaturation. Proteins that are resistant to digestion become more susceptible to digestion, independent of protein size, following thermal denaturation. For example, amino acid sequence coverage from digest fragments increases from 15 to 86% in myoglobin and from 0 to 43% in ovalbumin. This leads to more rapid and reliable protein identification by MALDI peptide mass mapping. Although some proteins aggregate upon thermal denaturation, the protein aggregates are easily digested by trypsin and generate sufficient numbers of digest fragments for protein identification.