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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) Number of members of the core pathways annotated byVogelsteinet al. showing significantly different expression. (B) The lists ofproteins with altered expression were mapped on to the Reactome PathwayDatabase and clustered into graph modules. (C) Principal componentanalysis of the quantified proteome shows that PC1 distinguishes hypermutatedfrom nonhypermutated cell lines. (D) The top contributors to PC1 amongthe Vogelstein gene set. SMAD4 and IDH2 are preferentially expressedin the hypermutated and nonhypermutated lines, respectively. (E,F)The Vogelstein gene set proteins with expression profiles most similarto SMAD4 (E) and IDH2 (F).
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fig5: (A) Number of members of the core pathways annotated byVogelsteinet al. showing significantly different expression. (B) The lists ofproteins with altered expression were mapped on to the Reactome PathwayDatabase and clustered into graph modules. (C) Principal componentanalysis of the quantified proteome shows that PC1 distinguishes hypermutatedfrom nonhypermutated cell lines. (D) The top contributors to PC1 amongthe Vogelstein gene set. SMAD4 and IDH2 are preferentially expressedin the hypermutated and nonhypermutated lines, respectively. (E,F)The Vogelstein gene set proteins with expression profiles most similarto SMAD4 (E) and IDH2 (F).

Mentions: A recent review by Vogelstein andcolleagues detailed a set of genes frequently mutated across manycancers and are thus likely to be drivers of the disease.34,35 The MultiNotch MS3 method detected many differentially expressedproteins in this gene set across several biological processes (Figure 5A). These included chromatin modification, PI3Kand RAS signaling, and the cell cycle. Mapping these proteins ontothe ReactomeFI network (Figure 5B)33 revealed four modules that correlate with knowncolorectal cancer biology. Notably, DNA mismatch repair defects area primary driver of mutational burden in heritable colorectal cancers.36 Similarly, signaling downstream of EGFR andWNT are often a critical drivers in many colorectal cancers.37


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) Number of members of the core pathways annotated byVogelsteinet al. showing significantly different expression. (B) The lists ofproteins with altered expression were mapped on to the Reactome PathwayDatabase and clustered into graph modules. (C) Principal componentanalysis of the quantified proteome shows that PC1 distinguishes hypermutatedfrom nonhypermutated cell lines. (D) The top contributors to PC1 amongthe Vogelstein gene set. SMAD4 and IDH2 are preferentially expressedin the hypermutated and nonhypermutated lines, respectively. (E,F)The Vogelstein gene set proteins with expression profiles most similarto SMAD4 (E) and IDH2 (F).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: (A) Number of members of the core pathways annotated byVogelsteinet al. showing significantly different expression. (B) The lists ofproteins with altered expression were mapped on to the Reactome PathwayDatabase and clustered into graph modules. (C) Principal componentanalysis of the quantified proteome shows that PC1 distinguishes hypermutatedfrom nonhypermutated cell lines. (D) The top contributors to PC1 amongthe Vogelstein gene set. SMAD4 and IDH2 are preferentially expressedin the hypermutated and nonhypermutated lines, respectively. (E,F)The Vogelstein gene set proteins with expression profiles most similarto SMAD4 (E) and IDH2 (F).
Mentions: A recent review by Vogelstein andcolleagues detailed a set of genes frequently mutated across manycancers and are thus likely to be drivers of the disease.34,35 The MultiNotch MS3 method detected many differentially expressedproteins in this gene set across several biological processes (Figure 5A). These included chromatin modification, PI3Kand RAS signaling, and the cell cycle. Mapping these proteins ontothe ReactomeFI network (Figure 5B)33 revealed four modules that correlate with knowncolorectal cancer biology. Notably, DNA mismatch repair defects area primary driver of mutational burden in heritable colorectal cancers.36 Similarly, signaling downstream of EGFR andWNT are often a critical drivers in many colorectal cancers.37

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