Quadrupole ion trap mass spectrometry: Current applications and future directions for peptide analysis

The capabilities of an ion trap mass spectrometer (ITMS) in determining the molecular weights and characterizing the primary sequences of peptides are explored. Ionization is accomplished by Cs+ desorption of the sample in a liquid matrix or by laser desorption from a solid matrix and is performed externally to the ion trap, which is operated with mass/charge range extension using resonance ejection. The mass spectra recorded in this way are dominated by the protonated molecules and are relatively free of the chemical noise normally associated with liquid secondary ion mass spectrometry (SIMS) or fast atom bombardment ionization. The matrix-assisted laser desorption spectra are of comparable quality to those obtained with Cs+ SIMS. The molecular weights of a variety of model peptides are measured with a resolution of 103 (FWHM) and a mass measurement accuracy of 0.1%, using mass analysis scan speeds in the 103–104 Da s−1 range. By slowing the scan speed by factors as large as 1000, better than unit resolution is achieved for all the compounds examined and, in the case of gramicidin S, a resolution of more than 105 has been recorded. Tandem mass spectrometry (MS/MS) is performed by isolating the protonated molecule in the trap using a reverse-then-forward scan and then irradiating it by resonance excitation to cause collision-induced dissociation. The product ion tandem mass spectrum is then recorded by the usual resonant ejection scan. Dissociation efficiencies are very high (>50%) and the product spectra are dominated by the fragments of amide bond scission together with associated CO losses (y, b and a-type fragments). In addition, internal cleavage products are also observed. Peptides such as prepro-VIP/PHM, renin substrate and glu-ribinopeptide give tandem spectra which are dominated by y ions, whereas other peptides, such as α-endorphin, give principally b-type fragment ions. The cyclic peptides, gramicidin S and the antibiotic actinomycin D, give spectra dominated by amide bond cleavages. Additional structural information, especially in the low-mass region, is sometimes desirable and can be provided by recording MS3 spectra. This can be done using little additional sample or analysis time. The high-resolution capabilities available by slowing the mass analysis scan time are applied to the MS/MS experiment, allowing unit resolution to be achieved, both in selecting the molecular ion and in recording the product ion spectrum. It is also shown that the higher-resolution resonant excitation experiment does not require isolation of the individual isotopic forms of the protonated molcule in order to provide an isotopically selective product spectrum. Sample sizes loaded onto the probe for MS/MS experiments are typically < 1 pmol and the data acquisition time is usually much shorter than the sample life-time. Shortcomings of the method include limitations common to desorption ionization (analyte suppression effects and reactions in the matrix) as well as the need to calibrate the mass scale with an external standard which results in mass measurement accuracies which, at present, are ±0.1%. A summary is given of steps which are being taken to improve mass measurement accuracy, to deposit increased internal energies to cause more extensive fragmentation, and to improve our understanding of ion traps through simulations of ion motion.