STEP (Statistical Test of Equivalent Pathways) analysis: a mass spectrometric method for carbohydrates and peptides.

We have recently developed a new mass spectrometry method, the STEP (statistical test of equivalent pathways) analysis that uses ion abundances in two tandem mass spectrometry experiments to obtain genealogy information about product ions present in mass spectra. The method requires minimal sample, and it can be performed using a conventional quadrupole ion trap mass spectrometer. To obtain genealogy information, STEP ratios are calculated by comparing the relative abundances of product ions in two MS/MS experiments. These ratios are directly related to the origin of the product ions. Product ions that result directly from the precursor ion always have STEP ratios that are </=1. Ions that result from secondary fragmentation pathways have STEP ratios that are significantly larger than the primary ions, based on a Q test of statistical significance. Consequently, the type (primary or secondary) of all the product ions in an MS/MS experiment can easily be identified in this analysis. The STEP method is applied herein to peptides and carbohydrates, and the STEP results are consistent with validation data for 95% of the ions in this study. This new method has many applications in carbohydrate and peptide analysis. It can be used to support mechanistic studies of peptide fragmentation, and it is useful for discriminating among various isomeric carbohydrates, without the need for reference standards. Several examples are presented to demonstrate the reliability of this method, and an example showing how the method benefits carbohydrate sequencing is also provided.