Development of a Peptide Mass Fingerprinting Method for Green Fluorescent Protein

Peptide mass fingerprinting (PMF) is a protein identification method in which a protein of interest is cleaved into small peptides using a digestive enzyme that cleaves at very specific peptide bonds. The peptides are then separated by high performance liquid chromatography (HPLC) and identified either through UV or mass detection. This study was conducted to create a PMF method for green fluorescent protein (GFP), and then to evaluate variations in the peptide map when the protein was subjected to forced degradation. A GFP digestion method was used involving heat denaturation, a buffer at an elevated pH, and the digestive enzyme, trypsin. The PMF method was developed and validated using HPLC and HPLC-MS. Following validation, degradation studies involving oxidation, hydrolysis, heat, and light were used to determine which peptides were targeted. For example, amino acids such as methionine, cysteine, histidine, tryptophan, and tyrosine are susceptible to oxidation. Theoretically, peptides incorporating these amino acid residues should be oxidized resulting in chromatographic peak shifts. These degradation results helped ensure the stability of GFP at the Biomanufacturing Training and Education Center (BTEC) at North Carolina State University. GFP serves as BTEC’s model therapeutic protein in their biomanufacturing process, and these results will help with the understanding of the degradation pathways of the protein. More detailed results will be presented.