Preferential Fragmentation of Protonated Gas-Phase Peptide Ions Adjacent to Acidic Amino Acid Residues

Matrix-assisted laser desorptiodionization (MALDI) is an effective method for producing protonated peptide and protein ions in the gas phase.' Together with electrospray ionization, MALDI has become an ionization method of choice for analyzing peptides and proteins by mass spectrometry (MS).2 The analyses are usually carried out by measuring the molecular masses of the intact polypeptides or peptide fragments produced by proteolysis or chemical degradation of proteins in solution. An alternative method for obtaining detailed primary structural information uses fragmentation of the protonated peptides in the gas phase followed by MS analysis of the fragment ions. A powerful variation of this approach, called tandem MS, employs mass spectrometric selection of the peptide ion of interest prior to fragmentation, followed by analysis of the fragment ion^.^^^ Fragmentation of peptide ions produced by MALDI has been studied in time-of-flight (TOF) mass analyzers (fitted with ion mirrors), instrumentation well-suited to the pulsed ionization ~ o u r c e . ~ * ~ Mass analyzers that utilize the principle of ion trapping are also well-suited to the pulsed MALDI ion s o ~ r c e . ~ Precursor ion selection can be achieved with high resolution.8 The prolonged time window of the measurement in the quadrupole ion trap to =1 s) compared with that in the ion mirror TOF analyzer (< s) enables the observation of fragmentation processes with low rate constants. In the reflecting TOF analyzer, highly preferential peptide bond cleavage has been observed at the bond carboxy (C)-terminal to aspartic acid residues, with Asp-Pro bonds exhibiting particularly high lability.6 In the present study, we investigate the fragmentation of MALDI-produced protonated peptide ions (containing an Arg residue and at least one acidic amino acid residue) in a quadrupole ion trap mass spectrometer and also observe preferential cleavage of the peptide bond adjacent to aspartic acid residues. In addition, we report the first observation of preferential fragmentation adjacent to glutamic acid residues. The investigation was carried out on a MALDI quadrupole ion trap mass spectrometer constructed at Rockefeller University? Ions are generated external to the ion trap using 355 nm laser irradiation,I0 accelerated to 60 eV, focused with an einzel lens, and injected axially into the trap. The trapped ions are