Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions

Abstract

By utilizing delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption/ionization (MALDI) technique, fast (< 320 ns) metastable ion fragmentation is observed for both peptide and protein analytes in the ion source of a linear time-of-flight mass spectrometer. Small peptides such as the oxidized B chain of bovine insulin exhibit fragmentation at the amide linking bond between peptide residues. Overlapping sequence information is provided by fragmentation from both the C- and N-terminal ends of the peptide (cn-, yn-, and z*n-type fragment ions). Larger proteins can also exhibit a wealth of sequence specific fragment ions in favorable cases. One example is cytochrome c, which undergoes substantial (approximately 80%) fast fragmentation at the amide bonds along the amino acid backbone of the protein. Only amide bond cleavages initiating from the C-terminal end (cn fragments) are observed. The observed fragmentation pattern provides a significant amount of potential sequence information for these molecules. External mass calibration of the intact protonated molecular ions is demonstrated with mass accuracies typically around 100 ppm. Mass accuracies for the observed fragment ions ranged from +/- 0.20 Da for the smaller peptides studied (i.e., oxidized B chain of bovine insulin) to +/- 0.38 Da for the largest protein studied (cytochrome c), based upon the known sequences.