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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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Quantitative performance.The 12-plex DiLeu-labeled yeast digestsamples were combined in 1:1 ratios across all channels and in 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d) and analyzed by LC–MS2 at 60k resolving power. Measured quantitative ratios of identifiedproteins (box and whiskers) are shown for (A) the 1:1 mixture in relationto neighboring channels and (B) the 16:1 mixture in relation to 16×channels (115a and 118d). Box plots demarcate the median (line), the25th and 75th percentile (box), and the 5th and 95th percentile (whiskers).
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fig4: Quantitative performance.The 12-plex DiLeu-labeled yeast digestsamples were combined in 1:1 ratios across all channels and in 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d) and analyzed by LC–MS2 at 60k resolving power. Measured quantitative ratios of identifiedproteins (box and whiskers) are shown for (A) the 1:1 mixture in relationto neighboring channels and (B) the 16:1 mixture in relation to 16×channels (115a and 118d). Box plots demarcate the median (line), the25th and 75th percentile (box), and the 5th and 95th percentile (whiskers).

Mentions: Next, we aimed to demonstratethe quantitative precision, accuracy,and dynamic range of the 12-plex DiLeu reagents for bottom-up proteinquantitation by labeling a complex mixture of S. cerevisiae lysate tryptic peptides and analyzing by high-resolution LC–MS2. Yeast lysate was digested with trypsin/Lys C, desalted,split into equal aliquots, and labeled in triplicate with each ofthe 12 DiLeu reagents. The 12-plex DiLeu-labeled yeast peptide sampleswere then prepared by combining at 1:1:1:1:1:1:1:1:1:1:1:1 and 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d). Samples were acquired on the Orbitrap Eliteusing a data-dependent top 10 method with HCD MS2 acquisitionat a resolving power of 60k. While 30k resolving power is sufficientfor baseline separation of the reporters, 60k resolving power waschosen for this experiment because it further resolves several interferingisotopic peaks from the surrounding reporters and allows for moreaccurate isotopic interference correction. Data from the triplicateLC–MS2 runs were combined in Proteome Discover tocalculate reporter ion ratios for 663 and 712 identified protein groupsfrom the 1:1 and 16:1 samples, respectively. After isotopic interference corrections were applied in Excel (Figure S-1 in the Supporting Information), the 12-plex DiLeu ratiosfor all quantified proteins were plotted against each other (Figure 4). Across all channels, the median ratios measurewithin 10% of the expected values with average coefficients of variation(CVs) of 7.9% for the 1:1 ratio sample and 11.5% for the 16:1 ratiosample. Reproducibility and variance of the protein quantitative ratiosbetween 16:1 replicates were compared and showed excellent correlationwith each other (Figure 5). To also characterizethe quantitative performance across the measured peptide dynamic rangewithin a sample, reporter ion ratios of PSMs were plotted as a functionof precursor ion signal intensity for a 12-plex DiLeu-labeled yeastlysate digest sample labeled in 10:1 ratios between neighboring channels(Figure S-3 in the Supporting Information). Variability of reporter ion ratios was fairly consistent acrossthe 5 orders of magnitude of precursor intensity. These results showthat the overall accuracy and precision remains excellent for highlymultiplexed, complex proteomics experiments across a usable dynamicrange. Furthermore, the increase in multiplexing also does not negativelyimpact peptide backbone fragmentation. An example HCD MS2 spectrum of a yeast lysate peptide yielding high coverage of b-and y-ions is shown (Figure 6).


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

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

Quantitative performance.The 12-plex DiLeu-labeled yeast digestsamples were combined in 1:1 ratios across all channels and in 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d) and analyzed by LC–MS2 at 60k resolving power. Measured quantitative ratios of identifiedproteins (box and whiskers) are shown for (A) the 1:1 mixture in relationto neighboring channels and (B) the 16:1 mixture in relation to 16×channels (115a and 118d). Box plots demarcate the median (line), the25th and 75th percentile (box), and the 5th and 95th percentile (whiskers).
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fig4: Quantitative performance.The 12-plex DiLeu-labeled yeast digestsamples were combined in 1:1 ratios across all channels and in 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d) and analyzed by LC–MS2 at 60k resolving power. Measured quantitative ratios of identifiedproteins (box and whiskers) are shown for (A) the 1:1 mixture in relationto neighboring channels and (B) the 16:1 mixture in relation to 16×channels (115a and 118d). Box plots demarcate the median (line), the25th and 75th percentile (box), and the 5th and 95th percentile (whiskers).
Mentions: Next, we aimed to demonstratethe quantitative precision, accuracy,and dynamic range of the 12-plex DiLeu reagents for bottom-up proteinquantitation by labeling a complex mixture of S. cerevisiae lysate tryptic peptides and analyzing by high-resolution LC–MS2. Yeast lysate was digested with trypsin/Lys C, desalted,split into equal aliquots, and labeled in triplicate with each ofthe 12 DiLeu reagents. The 12-plex DiLeu-labeled yeast peptide sampleswere then prepared by combining at 1:1:1:1:1:1:1:1:1:1:1:1 and 16:8:4:2:1:10:10:1:2:4:8:16ratios (115a–118d). Samples were acquired on the Orbitrap Eliteusing a data-dependent top 10 method with HCD MS2 acquisitionat a resolving power of 60k. While 30k resolving power is sufficientfor baseline separation of the reporters, 60k resolving power waschosen for this experiment because it further resolves several interferingisotopic peaks from the surrounding reporters and allows for moreaccurate isotopic interference correction. Data from the triplicateLC–MS2 runs were combined in Proteome Discover tocalculate reporter ion ratios for 663 and 712 identified protein groupsfrom the 1:1 and 16:1 samples, respectively. After isotopic interference corrections were applied in Excel (Figure S-1 in the Supporting Information), the 12-plex DiLeu ratiosfor all quantified proteins were plotted against each other (Figure 4). Across all channels, the median ratios measurewithin 10% of the expected values with average coefficients of variation(CVs) of 7.9% for the 1:1 ratio sample and 11.5% for the 16:1 ratiosample. Reproducibility and variance of the protein quantitative ratiosbetween 16:1 replicates were compared and showed excellent correlationwith each other (Figure 5). To also characterizethe quantitative performance across the measured peptide dynamic rangewithin a sample, reporter ion ratios of PSMs were plotted as a functionof precursor ion signal intensity for a 12-plex DiLeu-labeled yeastlysate digest sample labeled in 10:1 ratios between neighboring channels(Figure S-3 in the Supporting Information). Variability of reporter ion ratios was fairly consistent acrossthe 5 orders of magnitude of precursor intensity. These results showthat the overall accuracy and precision remains excellent for highlymultiplexed, complex proteomics experiments across a usable dynamicrange. Furthermore, the increase in multiplexing also does not negativelyimpact peptide backbone fragmentation. An example HCD MS2 spectrum of a yeast lysate peptide yielding high coverage of b-and y-ions is shown (Figure 6).

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