Application of the statistical test of equivalent pathways (STEP) method to the triple quadrupole mass spectrometer

Abstract

Recently, we demonstrated a new method, STEP (Statistical Test of Equivalent Pathways) analysis, which differentiates first-generation product ions (primary product ions) from second-generation product ions (secondary product ions) obtained in tandem mass spectrometric (MS/MS) experiments on a quadrupole ion trap mass spectrometer. The study presented here defines how to adapt the STEP method to a more routinely used mass analyzer, the triple quadrupole. New ion activation conditions were developed to adapt the STEP method to the triple quadrupole mass spectrometer using peptides and carbohydrates. The application of this method to the triple quadrupole is useful because it provides an efficient approach to differentiate primary and secondary ions on this instrument. Out of the total number of ions that were subjected to the STEP analysis, this method correctly identified 96% of ions as primary or secondary, indicating that this analysis is effective for carbohydrates and peptides undergoing collision-induced dissociation (CID) on a triple quadrupole mass spectrometer.The study of the nature of characteristic fragmentations of gaseous ions by mass spectrometry is important because understanding basic fragmentation information is helpful in deducing precursor ion structures for unknown compounds. 1-3 Fragmentation information can be used to establish genealogical relationships, which are important in discerning information about the connectivity of groups that fragment during tandem mass spectrometric (MS/MS) experiments. 4 Since first-generation product ions (primary product ions) directly originate from the precursor ion while the second generation of ions (secondary product ions) originate from other product ions in the MS/MS experiment, genealogy information can be useful in piecing together the structure of a precursor ion, based on the product ions detected in the MS/MS data. [5][6][7] The information about the connectivity of ions from MS/ MS data can be determined in a number of ways. For example, previous researchers have shown that some information can be inferred by comparing results in MS 3 experiments to MS 2 experiments on selected ions. 5 The product ions that appear on both the MS 2 and MS 3 data were assigned as a result of secondary fragmentation. However, the capability to perform MS n experiments is limited to trapping instruments like the quadrupole ion trap mass spectrometer and not possible in instruments like the triple quadrupole mass spectrometer. Nevertheless, Harrison and co-workers have used pseudo-MS 3 experiments to obtain genealogy information on a triple quadrupole mass spectrometer by performing collision-induced dissociation (CID) both in-source and in the collision cell. 8,9 Double-resonance experiments in conjunction with SWIFT (stored wave form inverse Fourier transform) techniques are another avenue for obtaining genealogy information in quadruple ion traps and Fourier transform ion cyclotron resonance mass spectrometers. 2,3,10,11 Application of the SWIFT technique allows p...