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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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Related in: MedlinePlus

The 12-plexDiLeu reporter ion peaks. The 12-plex reporters weremixed in a 1:1 ratio and infused directly into the Orbitrap Elitemass spectrometer, subjected to HCD MS2 fragmentation,and acquired at resolving powers 15–240k.
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fig3: The 12-plexDiLeu reporter ion peaks. The 12-plex reporters weremixed in a 1:1 ratio and infused directly into the Orbitrap Elitemass spectrometer, subjected to HCD MS2 fragmentation,and acquired at resolving powers 15–240k.

Mentions: In order to determine the resolvingpower at which the 12 reporterions could be baseline-resolved, we combined each of the 12 reportersat equal concentrations and infused the mixture into an Orbitrap Elitemass spectrometer using HCD MS2 acquisition in the Orbitrapat resolving powers ranging from 15k to 240k (at m/z 400) (Figure 3). At aresolving power of 15k, the separation between neighboring reportervariants is insufficient for accurate intensity measurements for quantitation.At a resolving power of 30k, reporters are baseline-resolved intounique peaks that are suitable for accurate quantitation. At a resolvingpower of 60k and greater, the −1 isotopic peaks from channels116c, 117b, 117c, 118b, and 118c are also baseline-resolved betweenthe surrounding reporters. Because these resolved isotopic peaks nolonger interfere with the surrounding primary reporter ion peaks at60k resolving power, the most accurate quantitation can be achievedat this resolution following isotopic interference correction.


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 peaks. The 12-plex reporters weremixed in a 1:1 ratio and infused directly into the Orbitrap Elitemass spectrometer, subjected to HCD MS2 fragmentation,and acquired at resolving powers 15–240k.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The 12-plexDiLeu reporter ion peaks. The 12-plex reporters weremixed in a 1:1 ratio and infused directly into the Orbitrap Elitemass spectrometer, subjected to HCD MS2 fragmentation,and acquired at resolving powers 15–240k.
Mentions: In order to determine the resolvingpower at which the 12 reporterions could be baseline-resolved, we combined each of the 12 reportersat equal concentrations and infused the mixture into an Orbitrap Elitemass spectrometer using HCD MS2 acquisition in the Orbitrapat resolving powers ranging from 15k to 240k (at m/z 400) (Figure 3). At aresolving power of 15k, the separation between neighboring reportervariants is insufficient for accurate intensity measurements for quantitation.At a resolving power of 30k, reporters are baseline-resolved intounique peaks that are suitable for accurate quantitation. At a resolvingpower of 60k and greater, the −1 isotopic peaks from channels116c, 117b, 117c, 118b, and 118c are also baseline-resolved betweenthe surrounding reporters. Because these resolved isotopic peaks nolonger interfere with the surrounding primary reporter ion peaks at60k resolving power, the most accurate quantitation can be achievedat this resolution following isotopic interference correction.

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