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High-resolution enabled 12-plex DiLeu isobaric tags for quantitative proteomics.

Frost DC, Greer T, Li L - Anal. Chem. (2014)

Bottom Line: In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group.The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation.We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Wisconsin , 777 Highland Avenue, Madison, Wisconsin 53705, United States.

ABSTRACT
Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.

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The 12-plexDiLeu reporter ion structures showing stable isotopepositions.
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fig2: The 12-plexDiLeu reporter ion structures showing stable isotopepositions.

Mentions: It was demonstrated recently with the TMT reagents that additionalisotopologues could be created by incorporation of 15Ninstead of 13C in the reporter groups. In doing so, thetwo reporter ions differ by 6.32 mDa, which can be baseline-resolvedusing high-resolution MSn acquisition.21,22 Given the similarities between TMT and DiLeu reagents, it stoodto reason that additional DiLeu reporters could be developed in asimilar manner using alternative combinations of 12C/13C, 14N/15N, and 1H/2H. Through calculated substitution of these isotopes in theDiLeu reporter structure, we designed eight new reporter isotopologueswith unique “pseudoisobaric” masses differing from theoriginal four by intervals of 5.84 mDa or 6.32 mDa to bring the totalnumber of reporters to 12 (Figure 2). The resulting12-plex set of isobaric DiLeu reagents is composed of two 115 variants,three 116 variants, three 117 variants, and four 118 variants (Figure 1B). In designing the new reporters, no syntheticsteps were added, and no custom isotopic reagents were needed.


High-resolution enabled 12-plex DiLeu isobaric tags for quantitative proteomics.

Frost DC, Greer T, Li L - Anal. Chem. (2014)

The 12-plexDiLeu reporter ion structures showing stable isotopepositions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4318621&req=5

fig2: The 12-plexDiLeu reporter ion structures showing stable isotopepositions.
Mentions: It was demonstrated recently with the TMT reagents that additionalisotopologues could be created by incorporation of 15Ninstead of 13C in the reporter groups. In doing so, thetwo reporter ions differ by 6.32 mDa, which can be baseline-resolvedusing high-resolution MSn acquisition.21,22 Given the similarities between TMT and DiLeu reagents, it stoodto reason that additional DiLeu reporters could be developed in asimilar manner using alternative combinations of 12C/13C, 14N/15N, and 1H/2H. Through calculated substitution of these isotopes in theDiLeu reporter structure, we designed eight new reporter isotopologueswith unique “pseudoisobaric” masses differing from theoriginal four by intervals of 5.84 mDa or 6.32 mDa to bring the totalnumber of reporters to 12 (Figure 2). The resulting12-plex set of isobaric DiLeu reagents is composed of two 115 variants,three 116 variants, three 117 variants, and four 118 variants (Figure 1B). In designing the new reporters, no syntheticsteps were added, and no custom isotopic reagents were needed.

Bottom Line: In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group.The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation.We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Wisconsin , 777 Highland Avenue, Madison, Wisconsin 53705, United States.

ABSTRACT
Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of (13)C, (15)N, and (2)H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography-tandem mass spectrometry (nanoLC-MS(2)) analysis on an Orbitrap Elite mass spectrometer.

Show MeSH
Related in: MedlinePlus