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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 general structure. (A) The DiLeu isobaric labelingreagent consists of a reporter group, balance group, and amine-reactivetriazine ester group. Stars indicate positions of isotopic substitution.(B) Stable isotopes (13C, 2H, and 15N) incorporated into the reporter group are mass-balanced by stableisotopes (13C, 18O) in the carbonyl balancegroup. Unique combinations of isotopes incorporated into the reportergroup yield two 115 variants, three 116 variants, three 117 variants,and four 118 variants whose isotopologues differ in mass by approximately6 mDa.
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fig1: The 12-plexDiLeu general structure. (A) The DiLeu isobaric labelingreagent consists of a reporter group, balance group, and amine-reactivetriazine ester group. Stars indicate positions of isotopic substitution.(B) Stable isotopes (13C, 2H, and 15N) incorporated into the reporter group are mass-balanced by stableisotopes (13C, 18O) in the carbonyl balancegroup. Unique combinations of isotopes incorporated into the reportergroup yield two 115 variants, three 116 variants, three 117 variants,and four 118 variants whose isotopologues differ in mass by approximately6 mDa.

Mentions: The structureof the DiLeu isobaric labeling reagent follows thatof other isobaric reagents in that it is composed of a reporter group,a balance group, and an amine-reactive group: an N,N-dimethylated leucine makes up the reporter andthe balance groups, and a triazine ester amine-reactive moiety enablesselective modification of peptide N-termini and lysine side chains(Figure 1A). The inspiration for using dimethylatedleucine as a reporter group began with our previous observation thatMS2 fragmentation of dimethylated peptides containing N-terminalleucine yielded the most intense immonium a1 ions comparedto other N-terminal amino acids. N-terminal dimethylation also providesthe added benefit of enhancing peptide fragmentation and aiding inde novo sequencing,32,33 and the compact size of dimethylatedleucine results in a modest nominal mass addition to the peptide of145 Da per label (Supporting Information Figure S-1). Consequently, the optimal collision energy requiredto produce both abundant reporter ions and a wealth of peptide backbonefragment ions during collision-induced MSn fragmentation of labeled peptides is lower than that which is requiredfor unlabeled peptides.34 This is in contrastto TMT-labeled peptides, which require higher collision energy toyield adequate reporter ion and peptide backbone fragment signalscompared to unlabeled peptides.35 Whilethe DiLeu reagents incorporate deuteriums into the reporter group,DiLeu-labeled peptides do not suffer from significant shifts in LCretention time between the labels.31 Bygrouping the deuteriums around the polar dimethylated amine functionalgroup, the probability of their interaction with reversed-phase stationaryphase is low—as opposed to the strongly favored interactionof the hydrophobic leucine side chain with the stationary phase—andthe deuterium effect is minimized, in agreement with previous research.36 The triazine ester was chosen as the amine-reactivegroup because it activates quickly (within 1 h), does not requirepurification prior to labeling, and labels amines with high efficiency.


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

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

The 12-plexDiLeu general structure. (A) The DiLeu isobaric labelingreagent consists of a reporter group, balance group, and amine-reactivetriazine ester group. Stars indicate positions of isotopic substitution.(B) Stable isotopes (13C, 2H, and 15N) incorporated into the reporter group are mass-balanced by stableisotopes (13C, 18O) in the carbonyl balancegroup. Unique combinations of isotopes incorporated into the reportergroup yield two 115 variants, three 116 variants, three 117 variants,and four 118 variants whose isotopologues differ in mass by approximately6 mDa.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The 12-plexDiLeu general structure. (A) The DiLeu isobaric labelingreagent consists of a reporter group, balance group, and amine-reactivetriazine ester group. Stars indicate positions of isotopic substitution.(B) Stable isotopes (13C, 2H, and 15N) incorporated into the reporter group are mass-balanced by stableisotopes (13C, 18O) in the carbonyl balancegroup. Unique combinations of isotopes incorporated into the reportergroup yield two 115 variants, three 116 variants, three 117 variants,and four 118 variants whose isotopologues differ in mass by approximately6 mDa.
Mentions: The structureof the DiLeu isobaric labeling reagent follows thatof other isobaric reagents in that it is composed of a reporter group,a balance group, and an amine-reactive group: an N,N-dimethylated leucine makes up the reporter andthe balance groups, and a triazine ester amine-reactive moiety enablesselective modification of peptide N-termini and lysine side chains(Figure 1A). The inspiration for using dimethylatedleucine as a reporter group began with our previous observation thatMS2 fragmentation of dimethylated peptides containing N-terminalleucine yielded the most intense immonium a1 ions comparedto other N-terminal amino acids. N-terminal dimethylation also providesthe added benefit of enhancing peptide fragmentation and aiding inde novo sequencing,32,33 and the compact size of dimethylatedleucine results in a modest nominal mass addition to the peptide of145 Da per label (Supporting Information Figure S-1). Consequently, the optimal collision energy requiredto produce both abundant reporter ions and a wealth of peptide backbonefragment ions during collision-induced MSn fragmentation of labeled peptides is lower than that which is requiredfor unlabeled peptides.34 This is in contrastto TMT-labeled peptides, which require higher collision energy toyield adequate reporter ion and peptide backbone fragment signalscompared to unlabeled peptides.35 Whilethe DiLeu reagents incorporate deuteriums into the reporter group,DiLeu-labeled peptides do not suffer from significant shifts in LCretention time between the labels.31 Bygrouping the deuteriums around the polar dimethylated amine functionalgroup, the probability of their interaction with reversed-phase stationaryphase is low—as opposed to the strongly favored interactionof the hydrophobic leucine side chain with the stationary phase—andthe deuterium effect is minimized, in agreement with previous research.36 The triazine ester was chosen as the amine-reactivegroup because it activates quickly (within 1 h), does not requirepurification prior to labeling, and labels amines with high efficiency.

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