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MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes.

McAlister GC, Nusinow DP, Jedrychowski MP, Wühr M, Huttlin EL, Erickson BK, Rad R, Haas W, Gygi SP - Anal. Chem. (2014)

Bottom Line: These methods, however, have a significant sensitivity penalty.By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements.Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

View Article: PubMed Central - PubMed

Affiliation: Harvard Medical School, Department of Cell Biology , Boston, Massachusetts 02115, United States.

ABSTRACT
Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8,378 proteins in union and 6,168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174,704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

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(A) Yeast was digestedwith LysC and labeled with TMT (10:4:1:1:4:10).That sample was combined with a TMT labeled HeLa sample (1:1:1:0:0:0).(B) A TMT-labeled, yeast peptide (NAAWLVFANK) was interrogated inback-to-back scans using (left spectrum) MS2, where the MS1 precursorwas fragmented using HCD. (Middle) MS3, where the MS1 precursor wasfragmented with CID, and a single MS2 product ion was isolated andfragmented using HCD. And, (right) MultiNotch MS3, where multipleMS2 product ions were simultaneously isolated and fragmented. (C)The precursor populations of the standard and MultiNotch MS3 scansused to generate the reporters above (middle and bottom spectra, respectively).For reference, we also include the ITMS2 spectrum prior to MS3 precursorisolation (top).
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fig1: (A) Yeast was digestedwith LysC and labeled with TMT (10:4:1:1:4:10).That sample was combined with a TMT labeled HeLa sample (1:1:1:0:0:0).(B) A TMT-labeled, yeast peptide (NAAWLVFANK) was interrogated inback-to-back scans using (left spectrum) MS2, where the MS1 precursorwas fragmented using HCD. (Middle) MS3, where the MS1 precursor wasfragmented with CID, and a single MS2 product ion was isolated andfragmented using HCD. And, (right) MultiNotch MS3, where multipleMS2 product ions were simultaneously isolated and fragmented. (C)The precursor populations of the standard and MultiNotch MS3 scansused to generate the reporters above (middle and bottom spectra, respectively).For reference, we also include the ITMS2 spectrum prior to MS3 precursorisolation (top).

Mentions: TheTMT reagents were dissolved in 40 μL of acetonitrile, and 10μL of the solution was added to 100 μg of peptides dissolvedin 100 μL of 50 mM HEPES (pH 8.5). After incubating for 1 hat room temperature (22 °C), the reaction was quenched by adding8 μL of 5% w/v hydroxylamine. Following labeling, the samplewas combined in desired ratios. Yeast aliquots were mixed at 10:4:1:1:4:10,and HeLa was mixed at 1:1:1:0:0:0 (Figure 1A). Those two samples were then mixed at a 1/1 w/w ratio and subjectedto C18 solid-phase extraction.


MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes.

McAlister GC, Nusinow DP, Jedrychowski MP, Wühr M, Huttlin EL, Erickson BK, Rad R, Haas W, Gygi SP - Anal. Chem. (2014)

(A) Yeast was digestedwith LysC and labeled with TMT (10:4:1:1:4:10).That sample was combined with a TMT labeled HeLa sample (1:1:1:0:0:0).(B) A TMT-labeled, yeast peptide (NAAWLVFANK) was interrogated inback-to-back scans using (left spectrum) MS2, where the MS1 precursorwas fragmented using HCD. (Middle) MS3, where the MS1 precursor wasfragmented with CID, and a single MS2 product ion was isolated andfragmented using HCD. And, (right) MultiNotch MS3, where multipleMS2 product ions were simultaneously isolated and fragmented. (C)The precursor populations of the standard and MultiNotch MS3 scansused to generate the reporters above (middle and bottom spectra, respectively).For reference, we also include the ITMS2 spectrum prior to MS3 precursorisolation (top).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215866&req=5

fig1: (A) Yeast was digestedwith LysC and labeled with TMT (10:4:1:1:4:10).That sample was combined with a TMT labeled HeLa sample (1:1:1:0:0:0).(B) A TMT-labeled, yeast peptide (NAAWLVFANK) was interrogated inback-to-back scans using (left spectrum) MS2, where the MS1 precursorwas fragmented using HCD. (Middle) MS3, where the MS1 precursor wasfragmented with CID, and a single MS2 product ion was isolated andfragmented using HCD. And, (right) MultiNotch MS3, where multipleMS2 product ions were simultaneously isolated and fragmented. (C)The precursor populations of the standard and MultiNotch MS3 scansused to generate the reporters above (middle and bottom spectra, respectively).For reference, we also include the ITMS2 spectrum prior to MS3 precursorisolation (top).
Mentions: TheTMT reagents were dissolved in 40 μL of acetonitrile, and 10μL of the solution was added to 100 μg of peptides dissolvedin 100 μL of 50 mM HEPES (pH 8.5). After incubating for 1 hat room temperature (22 °C), the reaction was quenched by adding8 μL of 5% w/v hydroxylamine. Following labeling, the samplewas combined in desired ratios. Yeast aliquots were mixed at 10:4:1:1:4:10,and HeLa was mixed at 1:1:1:0:0:0 (Figure 1A). Those two samples were then mixed at a 1/1 w/w ratio and subjectedto C18 solid-phase extraction.

Bottom Line: These methods, however, have a significant sensitivity penalty.By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements.Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

View Article: PubMed Central - PubMed

Affiliation: Harvard Medical School, Department of Cell Biology , Boston, Massachusetts 02115, United States.

ABSTRACT
Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8,378 proteins in union and 6,168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174,704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

Show MeSH
Related in: MedlinePlus