Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification
© Reilly et al; licensee BioMed Central Ltd. 2011
Received: 8 June 2010
Accepted: 17 January 2011
Published: 17 January 2011
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.
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 . 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 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 present that can be used for sequencing. These sequencing difficulties are further compounded when attempting to identify peptides in very complex samples.
We propose that stable isotope labelling can improve the certainty of peptide identity. Stable isotope labelling amino acids in cell culture (SILAC), where the "heavy" isotope (13C, 15N etc.) is used for quantification of proteins by MS, is now a well-established, safe and reproducible technique . The advantage of SILAC is that the only chemical difference is a single neutron that alters the atom and subsequently the amino acid mass, while not altering its biological relevance. There have also been significant advances in the complete incorporation of stable isotopes into multicellular organisms such as mice, with heavy isotope mouse feed now commercially available [6, 7]. Thus the scope of experiments that incorporate stable isotope labelling is broad and can range from simple experiments in prokaryotes through to complex experiments in genetically modified mice. In this study we demonstrate how stable isotope labelling can be used for the discovery and identification of peptide metabolites. In contrast to similar studies where the protein or biotherapeutic is modified by incorporation of a stable isotope [8, 9], we propose the inverse, where the entire matrix is modified instead of the target protein. Complete incorporation of, in our case, 15N into the matrix of interest allows discrimination between matrix-derived metabolites and peptides derived from the target protein.
In addition to validating peptide identity, differential labeling can be combined with triple quadrupole technology to enable targeted data acquisition, whether in the form of multiple reaction monitoring (MRM) or during precursor ion scanning. The former technique is extremely sensitive and is currently being exploited for quantitation of known compounds; however, MRM requires knowledge of the precursor mass of the analyte which in the case of metabolites is often unknown. In contrast, precursor ion scanning can be performed without prior knowledge of the precursor mass. Combining this with the knowledge of a specific and distinguishing fragment ion can enable the use of "information dependant acquisition" (IDA) to reveal the full MS/MS of target compounds. We describe how, by using a stable isotope labeled matrix, we are able to employ the precursor ion scan to detect molecules liberated from insulin. Here the shifted matrix results in the otherwise "normal" fragment ions of the insulin becoming the unique and distinguishing features that can be harnessed to trigger targeted MS/MS analysis.
List of low mass target ions in native and stable isotope labelled peptides
Native Immonium ion
Related native ions
15N Immonium ion
Apartic acid, D
Glutamic acid, E
Insulin tryptic peptides
Target ions used for precursor scanning
120 (F), 70 (P/R/N), 86 (L/I), 72 (V)
120 (F), 70 (P/R/N)
72 (V), 86 (L/I), 70 (P/R/N)
Furthermore, the selection of the internal residues valine and phenylalanine illustrates the strength of the approach through its ability to employ a variety of low mass ions that can come from residues found throughout the target peptide.
Lastly this targeted approach to metabolite identification is especially relevant to the monitoring and discovery of biotherapeutic metabolites in complex matrices where targets are of unknown precursor mass. Here we took peptides derived from a tryptic digest of injectable insulin and added them to the same complex matrix mentioned above (40 μg 15N as above). We observed specificity and selectivity for peptides liberated from insulin by virtue of proteolysis by bacterial peptidases contained within the E. Coli lysate. The same sample was also run as a standard LC-MS/MS experiment and both results submitted to a ProteinPilot version 4 search algorithm. While the LC-MS/MS came back with no positive identifications for insulin, the precursor scan identified the predicted tryptic peptides as well as additional unexpected insulin peptides. These included peptides with modified cysteine residues and truncated peptides that were the result of residual protease activity within the 15N E. coli matrix.
Sequencing ions for the insulin peptide FVNQHLCGSHLVEALYLVCGER.
We have shown that by using stable isotope labeled matrices combined with precursor ion scanning and monitoring low mass ions, we are able to selectively screen for peptides liberated from proteins of interest. The precursor scanning technique assumes no prior knowledge or prediction of how the products within that matrix will form and is able to specifically screen peptides that may even contain unanticipated post translational modifications. The selectivity employed in triple quadrupole technology allows for targeted fragmentation data acquisition for the peptides/metabolites of interest at low abundance relative to a complex endogenous background. Furthermore, the data acquired is readily searchable using current informatics algorithms without a requirement to analyze MS/MS data containing 15N or alternative stable isotopes. This technique will find many applications in monitoring changes to biomolecules - especially in relation to biotherapeutics - within complex sample mixtures. Moreover, with the advent of isotopically labeled animal feed one can envision the use of this technique for absorption, distribution, metabolism, and excretion (ADME) type studies of peptide and protein drugs in experimental animals.
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