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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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Related in: MedlinePlus

(A) While infusing anion with an m/z ratio of 989, wevaried the isolation notch width and location.We recorded the isolation efficiency as a function of those parameters.This analysis was repeated for the series of ions, and the resultingdata set was fitted using linear regression. (B) During a 90 min MultiNotchLC–MS2/MS3 analysis of the yeast/human two-proteome sample,we isolated the MS3 precursor population without any subsequent fragmentation.We then calculated the fraction of MS2 ions retained in the MS3 precursorpopulation.
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fig2: (A) While infusing anion with an m/z ratio of 989, wevaried the isolation notch width and location.We recorded the isolation efficiency as a function of those parameters.This analysis was repeated for the series of ions, and the resultingdata set was fitted using linear regression. (B) During a 90 min MultiNotchLC–MS2/MS3 analysis of the yeast/human two-proteome sample,we isolated the MS3 precursor population without any subsequent fragmentation.We then calculated the fraction of MS2 ions retained in the MS3 precursorpopulation.

Mentions: All of the initial developmentwork for this method was focused on defining equations that couldaccurately describe the size and location for any possible notch inthe SPS waveform. To this end, we injected mixtures of ions with known m/z values into the instrument, and weisolated those ions using a series of SPS isolation waveforms. Duringthese experiments, we varied the notch width and location, and theisolation efficiency was recorded as a function of those parameters.Figure 2A shows the data generated by performingthis analysis on an ion with 989 m/z. As the q-value of the ion varies, so does theoptimal position and width of the isolation notch. This type of analysiswas repeated for many ions, and the resulting aggregate data setswere fitted using linear regression (Figure S2 in the Supporting Information).


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) While infusing anion with an m/z ratio of 989, wevaried the isolation notch width and location.We recorded the isolation efficiency as a function of those parameters.This analysis was repeated for the series of ions, and the resultingdata set was fitted using linear regression. (B) During a 90 min MultiNotchLC–MS2/MS3 analysis of the yeast/human two-proteome sample,we isolated the MS3 precursor population without any subsequent fragmentation.We then calculated the fraction of MS2 ions retained in the MS3 precursorpopulation.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: (A) While infusing anion with an m/z ratio of 989, wevaried the isolation notch width and location.We recorded the isolation efficiency as a function of those parameters.This analysis was repeated for the series of ions, and the resultingdata set was fitted using linear regression. (B) During a 90 min MultiNotchLC–MS2/MS3 analysis of the yeast/human two-proteome sample,we isolated the MS3 precursor population without any subsequent fragmentation.We then calculated the fraction of MS2 ions retained in the MS3 precursorpopulation.
Mentions: All of the initial developmentwork for this method was focused on defining equations that couldaccurately describe the size and location for any possible notch inthe SPS waveform. To this end, we injected mixtures of ions with known m/z values into the instrument, and weisolated those ions using a series of SPS isolation waveforms. Duringthese experiments, we varied the notch width and location, and theisolation efficiency was recorded as a function of those parameters.Figure 2A shows the data generated by performingthis analysis on an ion with 989 m/z. As the q-value of the ion varies, so does theoptimal position and width of the isolation notch. This type of analysiswas repeated for many ions, and the resulting aggregate data setswere fitted using linear regression (Figure S2 in the Supporting Information).

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