Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification
- PDF / 510,055 Bytes
- 8 Pages / 595.276 x 793.701 pts Page_size
- 65 Downloads / 137 Views
METHODOLOGY
Open Access
Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification Charles B Reilly, Sri H Ramarathinam, Nicholas A Williamson, Anthony W Purcell*
Abstract We describe a method to identify metabolites of proteins that eliminates endogenous background by using stable isotope labeled matrices. This technique allows selective screening of the intact therapeutic molecule and all metabolites using a modified precursor ion scan that monitors low molecular weight fragment ions produced during MS/MS. This distinct set of low mass ions differs between isotopically labeled and natural isotope containing species allowing excellent discrimination between endogenous compounds and target analytes. All compounds containing amino acids that consist of naturally abundant isotopes can be selected using this scanning technique for further analysis, including metabolites of the parent molecule. The sensitivity and selectivity of this technique is discussed with specific examples of insulin metabolites identified within a complex matrix using a range of different validated low mass target ions. Background Mass spectrometry (MS) can identify molecules based on their distinctive mass and/or on the mass of product ions generated by fragmentation of the parent ion. It is this sensitivity and selectivity that is often exploited to distinguish a target peptide from a mixture of other closely related species [1]. The common approach for peptide identification is to use characteristic fragment ions produced during relatively mild collision activated dissociation of parent ions. The series of “sequencing” ions produced differ in mass by the residue mass of specific amino acids in addition to various neutral losses and intramolecular reactions. These ions then enable the de novo reconstruction of the primary structure of the parent peptide ion from the spectrum of fragment ions. This systematic approach relies on the prediction the fragmentation pattern of the peptide, however many variables such as collision energy and sequence composition influence fragmentation, often making it difficult to predict ion intensity relationships. This can then lead to challenges in the interpretation and prediction of MS/MS spectrum [2-5]. The uncertainty in how a * Correspondence: [email protected] Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010 Australia
particular peptide may fragment and the reliance of a classical “sequencing ions” approach to peptide identification may in some cases lead to the incomplete assignment of the spectra, where sequencing ions provide an internal sequence “tag” but fail to provide enough information for a complete peptide sequence. This is especially the case when the data from which peptide identities are made is of poor quality due to very low abundance of the parent species, or if there is only a small number of MS/MS ions prese
Data Loading...
