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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) Eight colorectal cancer cell lines weregrown in biologicaltriplicate. Each replicate was digested with LysC, labeled with TMT,fractionated, and analyzed using MultiNotch MS3 (3-h LC gradients).(B) All protein ratios from replicates 1 and 2 were plotted againsteach other. In total this represents 172 704 quantitative ratios.(C) Across the three replicate we performed a one way ANOVA with Welch’scorrection. (D) We highlighted the protein expression profile fortwo commonly studied proteins, EGFR and MSH6, and (E) the WT and mutant(G13D) forms of KRAS.
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fig4: (A) Eight colorectal cancer cell lines weregrown in biologicaltriplicate. Each replicate was digested with LysC, labeled with TMT,fractionated, and analyzed using MultiNotch MS3 (3-h LC gradients).(B) All protein ratios from replicates 1 and 2 were plotted againsteach other. In total this represents 172 704 quantitative ratios.(C) Across the three replicate we performed a one way ANOVA with Welch’scorrection. (D) We highlighted the protein expression profile fortwo commonly studied proteins, EGFR and MSH6, and (E) the WT and mutant(G13D) forms of KRAS.

Mentions: Beyond demonstrating the technicalcapabilities of the MultiNotchMS3 method with the two-proteome model, we sought to demonstrate thepracticality of the method using a large-scale proteomics experiment.To this end, we prepared a TMT 8-plex sample that consisted of eightdifferent colorectal cancer cell lines: Colo-205, LoVo, DLD-1, SW48,HT-29, HCT-15, HT-55, and HCT-116 (Figure 4A). We grew all eight cell lines in biological triplicate, harvestedthe proteins, and digested the resulting proteome samples with LysC.Following digestion, we labeled the samples with the TMT reagents,mixed the labeled peptides, and fractionated the mixtures using offlinebasic-pH reverse phase HPLC. We collected 24 fractions, which we thenanalyzed using a 3-h LC–MS3 method. To analyze each biologicalreplicate required 3 days of analysis time, and to collect the entiredata set required 9 days.


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) Eight colorectal cancer cell lines weregrown in biologicaltriplicate. Each replicate was digested with LysC, labeled with TMT,fractionated, and analyzed using MultiNotch MS3 (3-h LC gradients).(B) All protein ratios from replicates 1 and 2 were plotted againsteach other. In total this represents 172 704 quantitative ratios.(C) Across the three replicate we performed a one way ANOVA with Welch’scorrection. (D) We highlighted the protein expression profile fortwo commonly studied proteins, EGFR and MSH6, and (E) the WT and mutant(G13D) forms of KRAS.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: (A) Eight colorectal cancer cell lines weregrown in biologicaltriplicate. Each replicate was digested with LysC, labeled with TMT,fractionated, and analyzed using MultiNotch MS3 (3-h LC gradients).(B) All protein ratios from replicates 1 and 2 were plotted againsteach other. In total this represents 172 704 quantitative ratios.(C) Across the three replicate we performed a one way ANOVA with Welch’scorrection. (D) We highlighted the protein expression profile fortwo commonly studied proteins, EGFR and MSH6, and (E) the WT and mutant(G13D) forms of KRAS.
Mentions: Beyond demonstrating the technicalcapabilities of the MultiNotchMS3 method with the two-proteome model, we sought to demonstrate thepracticality of the method using a large-scale proteomics experiment.To this end, we prepared a TMT 8-plex sample that consisted of eightdifferent colorectal cancer cell lines: Colo-205, LoVo, DLD-1, SW48,HT-29, HCT-15, HT-55, and HCT-116 (Figure 4A). We grew all eight cell lines in biological triplicate, harvestedthe proteins, and digested the resulting proteome samples with LysC.Following digestion, we labeled the samples with the TMT reagents,mixed the labeled peptides, and fractionated the mixtures using offlinebasic-pH reverse phase HPLC. We collected 24 fractions, which we thenanalyzed using a 3-h LC–MS3 method. To analyze each biologicalreplicate required 3 days of analysis time, and to collect the entiredata set required 9 days.

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