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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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Multiplexing comparison.DiLeu-labeled yeast lysate digest sampleswere prepared as 4-, 8-, and 12-plex mixtures in 10:1 ratios betweenneighboring channels and analyzed by LC–MS2 at 60kresolving power. Measured quantitative ratios of identified proteins(box and whiskers) are shown. Box plots demarcate the median (line),the 25th and 75th percentile (box), and the 5th and 95th percentile(whiskers).
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fig7: Multiplexing comparison.DiLeu-labeled yeast lysate digest sampleswere prepared as 4-, 8-, and 12-plex mixtures in 10:1 ratios betweenneighboring channels and analyzed by LC–MS2 at 60kresolving power. Measured quantitative ratios of identified proteins(box and whiskers) are shown. Box plots demarcate the median (line),the 25th and 75th percentile (box), and the 5th and 95th percentile(whiskers).

Mentions: We also explored the effect ofmultiplexing on peptide and proteinidentification and quantitative precision. Three DiLeu-labeled yeastpeptide samples were prepared: (1) a 4-plex mixture of 115b, 116a,117c, and 118d combined at a 10:1:10:1 ratio; (2) an 8-plex mixtureof 115a, 115b, 116b, 116c, 117a, 117b, 118c, and 118d combined ata 10:1:10:1:10:1:10:1 ratio; (3) a 12-plex mixture of 115a through118d combined at a 10:1:10:1:10:1:10:1:10:1:10:1 ratio. The 4-plexsample contained channels separated by 1 Da while the 8-plex containsfour pairs of channels separated by ∼6 mDa, providing a goodindication of the impact of increasing the multiplexing with closelyspaced reporter isotopologues. Peptide concentration and injectionvolume were equal across the three samples. Overall on-column sampleload was greater than that used for the 1:1 and 16:1 experiments.Samples were acquired in triplicate on the Orbitrap Elite using adata-dependent top 10 method with HCD MS2 acquisition ata resolving power of 60k (at 400 m/z). We chose to keep the resolving power and acquisition speed constantfor all three experiments in order to evaluate only the effect ofincreasing multiplexing with closely spaced reporters on identificationrates and quantitative precision. As such, the 4-plex experiment islimited by the slower MS2 acquisition speed at 60k resolvingpower. In practice, a typical 4-plex experiment with 1 Da-spaced reporterswould be acquired at the lowest MS2 resolution to achievethe fastest acquisition speed, yielding significantly more MS2 spectra and greater numbers of identified PSMs, peptides,and proteins than we observe in this comparison. Across triplicateruns, the 4-, 8-, and 12-plex experiments resulted in 1116, 1008,and 985 identified protein groups, respectively, and 5451, 4874, and4437 identified peptides, respectively. This represents a 12% reductionin protein identification rate and a 19% reduction in peptide identificationfor the 12-plex experiment. The reporter ratios of quantified proteinsfrom neighboring 10:1 channels were then plotted against each other(Figure 7). The 8-plex and 12-plex distributionsare broader than the 4-plex distributions, and median values deviateby varying degrees from the expected value. Average CVs for the proteinratios of the 4-, 8-, and 12-plex were 9.9%, 16.2%, and 14.2%, respectively;average CVs for PSM reporter ion ratios were 18.7%, 31.6%, and 28.0%,respectively. While the reductions in protein and peptide identificationrate and in quantitative accuracy and precision are not insignificant,we feel that these concessions are acceptable given the increase inanalytical throughput.


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

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

Multiplexing comparison.DiLeu-labeled yeast lysate digest sampleswere prepared as 4-, 8-, and 12-plex mixtures in 10:1 ratios betweenneighboring channels and analyzed by LC–MS2 at 60kresolving power. Measured quantitative ratios of identified proteins(box and whiskers) are shown. Box plots demarcate the median (line),the 25th and 75th percentile (box), and the 5th and 95th percentile(whiskers).
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Related In: Results  -  Collection

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fig7: Multiplexing comparison.DiLeu-labeled yeast lysate digest sampleswere prepared as 4-, 8-, and 12-plex mixtures in 10:1 ratios betweenneighboring channels and analyzed by LC–MS2 at 60kresolving power. Measured quantitative ratios of identified proteins(box and whiskers) are shown. Box plots demarcate the median (line),the 25th and 75th percentile (box), and the 5th and 95th percentile(whiskers).
Mentions: We also explored the effect ofmultiplexing on peptide and proteinidentification and quantitative precision. Three DiLeu-labeled yeastpeptide samples were prepared: (1) a 4-plex mixture of 115b, 116a,117c, and 118d combined at a 10:1:10:1 ratio; (2) an 8-plex mixtureof 115a, 115b, 116b, 116c, 117a, 117b, 118c, and 118d combined ata 10:1:10:1:10:1:10:1 ratio; (3) a 12-plex mixture of 115a through118d combined at a 10:1:10:1:10:1:10:1:10:1:10:1 ratio. The 4-plexsample contained channels separated by 1 Da while the 8-plex containsfour pairs of channels separated by ∼6 mDa, providing a goodindication of the impact of increasing the multiplexing with closelyspaced reporter isotopologues. Peptide concentration and injectionvolume were equal across the three samples. Overall on-column sampleload was greater than that used for the 1:1 and 16:1 experiments.Samples were acquired in triplicate on the Orbitrap Elite using adata-dependent top 10 method with HCD MS2 acquisition ata resolving power of 60k (at 400 m/z). We chose to keep the resolving power and acquisition speed constantfor all three experiments in order to evaluate only the effect ofincreasing multiplexing with closely spaced reporters on identificationrates and quantitative precision. As such, the 4-plex experiment islimited by the slower MS2 acquisition speed at 60k resolvingpower. In practice, a typical 4-plex experiment with 1 Da-spaced reporterswould be acquired at the lowest MS2 resolution to achievethe fastest acquisition speed, yielding significantly more MS2 spectra and greater numbers of identified PSMs, peptides,and proteins than we observe in this comparison. Across triplicateruns, the 4-, 8-, and 12-plex experiments resulted in 1116, 1008,and 985 identified protein groups, respectively, and 5451, 4874, and4437 identified peptides, respectively. This represents a 12% reductionin protein identification rate and a 19% reduction in peptide identificationfor the 12-plex experiment. The reporter ratios of quantified proteinsfrom neighboring 10:1 channels were then plotted against each other(Figure 7). The 8-plex and 12-plex distributionsare broader than the 4-plex distributions, and median values deviateby varying degrees from the expected value. Average CVs for the proteinratios of the 4-, 8-, and 12-plex were 9.9%, 16.2%, and 14.2%, respectively;average CVs for PSM reporter ion ratios were 18.7%, 31.6%, and 28.0%,respectively. While the reductions in protein and peptide identificationrate and in quantitative accuracy and precision are not insignificant,we feel that these concessions are acceptable given the increase inanalytical throughput.

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