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Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification.

Reilly CB, Ramarathinam SH, Williamson NA, Purcell AW - Proteome Sci (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010 Australia. apurcell@unimelb.edu.au.

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.

No MeSH data available.


Insulin peptides were combined with a 15N complex matrix. 2 pmol of an insulin tryptic digest was added to 40 μg (total protein) of a 15N matrix, consisting of the acid soluble fraction of E. coli. The same sample was run as a regular LC-MSMS experiment and as precursor ion scanning experiments. Target ions during the precursor scans consisted of immonium and related ions corresponding to residues L, I, Y, V, N, D and F. (A) The base peak chromatogram during the LC-MS/MS procedure. (B) The total ion chromatogram (TIC) for the precursor ion scan with target ion 86 m/z which corresponds to Ile and Leu. (C) The precursor scan at time point 50.6 min (which corresponds to the major peak in the TIC) showing the presence of an intense peak for 651.5 m/z which corresponds to the insulin peptide FVNQHLCGSHLVEALYLVCGER. (D) The mass spectrum at the 50.6 min time point showing that the 651.5 m/z ion is of low abundance relative to the complex spectral background. (E) The Product ion spectrum for the ion at m/z 651.5 which was acquired due to detection of a target ion during the precursor scan at time point 50.6 min. The sequencing ions lead to a positive identification of the peptide FVNQHLCGSHLVEALYLVCGER. Both the LC-MS/MS and precursor ion scan data sets were submitted to the same ProteinPilot version 4 search engine. Only the Precursor ion scan identified peptides liberated from insulin.
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Figure 3: Insulin peptides were combined with a 15N complex matrix. 2 pmol of an insulin tryptic digest was added to 40 μg (total protein) of a 15N matrix, consisting of the acid soluble fraction of E. coli. The same sample was run as a regular LC-MSMS experiment and as precursor ion scanning experiments. Target ions during the precursor scans consisted of immonium and related ions corresponding to residues L, I, Y, V, N, D and F. (A) The base peak chromatogram during the LC-MS/MS procedure. (B) The total ion chromatogram (TIC) for the precursor ion scan with target ion 86 m/z which corresponds to Ile and Leu. (C) The precursor scan at time point 50.6 min (which corresponds to the major peak in the TIC) showing the presence of an intense peak for 651.5 m/z which corresponds to the insulin peptide FVNQHLCGSHLVEALYLVCGER. (D) The mass spectrum at the 50.6 min time point showing that the 651.5 m/z ion is of low abundance relative to the complex spectral background. (E) The Product ion spectrum for the ion at m/z 651.5 which was acquired due to detection of a target ion during the precursor scan at time point 50.6 min. The sequencing ions lead to a positive identification of the peptide FVNQHLCGSHLVEALYLVCGER. Both the LC-MS/MS and precursor ion scan data sets were submitted to the same ProteinPilot version 4 search engine. Only the Precursor ion scan identified peptides liberated from insulin.

Mentions: The experimental cycle loop of the 4000 QTrap instrument used consisted of precursor ion scans that targeted up to four specific immonium ions, with the detection of any one of these ions prompting IDA. This in turn resulted in acquisition of an enhanced product ion spectrum and an EMS. Figure 3 shows a total ion chromatogram (TIC) for the precursor scan for m/z 86 (immonium ion corresponding to leucine/isoleucine) and a major peak at time point 50.6 min. At this time point the major precursor mass that was giving a signal for the presence of an 86 m/z was 651.7 and accordingly an IDA-triggered EPI was generated. The MS/MS data in this spectrum corresponds to that of the insulin-derived tryptic-like peptide FVNQHLCGSHLVEALYLVCGER (Table 2). In addition to the EPI we included an EMS to be triggered during the precursor scan in order to provide context. Figure 3 (bottom right panel) shows the mass spectrum at the same time point for when the 651 m/z peptide was identified (50.6 min). Here we see that the peptide would not have been observed during a conventional MS/MS acquisition due to the large number of ions with much greater ion intensity than the insulin peptide signal. These data shows that despite the target peptide being in low abundance relative to the complex spectral background it still gives the highest intensity signal during the precursor scan, resulting in "targeted data acquisition" for a peptide that was derived from insulin regardless of matrix complexity. When the data acquired was subjected to analysis by ProteinPilot version 4 the only successful protein ID was insulin.


Stable isotope shifted matrices enable the use of low mass ion precursor scanning for targeted metabolite identification.

Reilly CB, Ramarathinam SH, Williamson NA, Purcell AW - Proteome Sci (2011)

Insulin peptides were combined with a 15N complex matrix. 2 pmol of an insulin tryptic digest was added to 40 μg (total protein) of a 15N matrix, consisting of the acid soluble fraction of E. coli. The same sample was run as a regular LC-MSMS experiment and as precursor ion scanning experiments. Target ions during the precursor scans consisted of immonium and related ions corresponding to residues L, I, Y, V, N, D and F. (A) The base peak chromatogram during the LC-MS/MS procedure. (B) The total ion chromatogram (TIC) for the precursor ion scan with target ion 86 m/z which corresponds to Ile and Leu. (C) The precursor scan at time point 50.6 min (which corresponds to the major peak in the TIC) showing the presence of an intense peak for 651.5 m/z which corresponds to the insulin peptide FVNQHLCGSHLVEALYLVCGER. (D) The mass spectrum at the 50.6 min time point showing that the 651.5 m/z ion is of low abundance relative to the complex spectral background. (E) The Product ion spectrum for the ion at m/z 651.5 which was acquired due to detection of a target ion during the precursor scan at time point 50.6 min. The sequencing ions lead to a positive identification of the peptide FVNQHLCGSHLVEALYLVCGER. Both the LC-MS/MS and precursor ion scan data sets were submitted to the same ProteinPilot version 4 search engine. Only the Precursor ion scan identified peptides liberated from insulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Insulin peptides were combined with a 15N complex matrix. 2 pmol of an insulin tryptic digest was added to 40 μg (total protein) of a 15N matrix, consisting of the acid soluble fraction of E. coli. The same sample was run as a regular LC-MSMS experiment and as precursor ion scanning experiments. Target ions during the precursor scans consisted of immonium and related ions corresponding to residues L, I, Y, V, N, D and F. (A) The base peak chromatogram during the LC-MS/MS procedure. (B) The total ion chromatogram (TIC) for the precursor ion scan with target ion 86 m/z which corresponds to Ile and Leu. (C) The precursor scan at time point 50.6 min (which corresponds to the major peak in the TIC) showing the presence of an intense peak for 651.5 m/z which corresponds to the insulin peptide FVNQHLCGSHLVEALYLVCGER. (D) The mass spectrum at the 50.6 min time point showing that the 651.5 m/z ion is of low abundance relative to the complex spectral background. (E) The Product ion spectrum for the ion at m/z 651.5 which was acquired due to detection of a target ion during the precursor scan at time point 50.6 min. The sequencing ions lead to a positive identification of the peptide FVNQHLCGSHLVEALYLVCGER. Both the LC-MS/MS and precursor ion scan data sets were submitted to the same ProteinPilot version 4 search engine. Only the Precursor ion scan identified peptides liberated from insulin.
Mentions: The experimental cycle loop of the 4000 QTrap instrument used consisted of precursor ion scans that targeted up to four specific immonium ions, with the detection of any one of these ions prompting IDA. This in turn resulted in acquisition of an enhanced product ion spectrum and an EMS. Figure 3 shows a total ion chromatogram (TIC) for the precursor scan for m/z 86 (immonium ion corresponding to leucine/isoleucine) and a major peak at time point 50.6 min. At this time point the major precursor mass that was giving a signal for the presence of an 86 m/z was 651.7 and accordingly an IDA-triggered EPI was generated. The MS/MS data in this spectrum corresponds to that of the insulin-derived tryptic-like peptide FVNQHLCGSHLVEALYLVCGER (Table 2). In addition to the EPI we included an EMS to be triggered during the precursor scan in order to provide context. Figure 3 (bottom right panel) shows the mass spectrum at the same time point for when the 651 m/z peptide was identified (50.6 min). Here we see that the peptide would not have been observed during a conventional MS/MS acquisition due to the large number of ions with much greater ion intensity than the insulin peptide signal. These data shows that despite the target peptide being in low abundance relative to the complex spectral background it still gives the highest intensity signal during the precursor scan, resulting in "targeted data acquisition" for a peptide that was derived from insulin regardless of matrix complexity. When the data acquired was subjected to analysis by ProteinPilot version 4 the only successful protein ID was insulin.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010 Australia. apurcell@unimelb.edu.au.

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.

No MeSH data available.