2014
DOI: 10.1021/ac502040v
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MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

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 (PT… Show more

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Cited by 1,200 publications
(1,389 citation statements)
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“…S1) prompted us to investigate their abundance at the protein level. To do so, we performed global quantitative proteomics in BAT of cold-acclimated WT and UCP1-KO mice using isobaric tagging (18), which provided quantitation of 6,354 proteins (Dataset S1). Unsupervised k-means clustering of the proteomic data revealed a total of six clusters (Datasets S2-S7), two of which (clusters 4 and 5) were robustly distinct between WT and UCP1-KO BAT (Fig.…”
Section: Significancementioning
confidence: 99%
“…S1) prompted us to investigate their abundance at the protein level. To do so, we performed global quantitative proteomics in BAT of cold-acclimated WT and UCP1-KO mice using isobaric tagging (18), which provided quantitation of 6,354 proteins (Dataset S1). Unsupervised k-means clustering of the proteomic data revealed a total of six clusters (Datasets S2-S7), two of which (clusters 4 and 5) were robustly distinct between WT and UCP1-KO BAT (Fig.…”
Section: Significancementioning
confidence: 99%
“…Other differential proteomics techniques, including stable isotope labeling by amino acids in cell culture (Lewandowska et al, 2013), hydroponic isotope labeling of entire plants (Bindschedler et al, 2008), 13 CO 2 labeling (Chen et al, 2011), and isobaric tags for relative and absolute quantitation (Ge et al, 2013), have emerged in recent years as tools to probe in vivo protein synthesis in plants. Notably, multiplexed quantifications with techniques such as isobaric tags for relative and absolute quantitation are inherently problematic due to known interference problems when using ion trap sources (McAlister et al, 2014). Modern instruments with MultiNotch MS3 capabilities can overcome these limitations but impose significant sensitivity penalties (McAlister et al, 2014).…”
mentioning
confidence: 99%
“…Notably, multiplexed quantifications with techniques such as isobaric tags for relative and absolute quantitation are inherently problematic due to known interference problems when using ion trap sources (McAlister et al, 2014). Modern instruments with MultiNotch MS3 capabilities can overcome these limitations but impose significant sensitivity penalties (McAlister et al, 2014). The instrumentation required for such sophisticated experiments is expensive and limits widespread use.…”
mentioning
confidence: 99%
“…Specifically, the reporter ion intensity of each sample was divided by the sum of all reporter ion intensities and the calculated ratio was multiplied with the corresponding MS1 protein intensity. In this way, sample specific MS1 intensities and iBAQ values could be calculated and absolute protein concentrations estimated in each of the 6 samples as recently specified [25,38,41]. In detail, the MS1 intensities were divided by the number of possible tryptic peptides amenable for LC--MS analysis of each protein to control protein size and sequence differences and log transformed.…”
Section: Estimation Of Cellular Protein Abundances (Tmt Dataset)mentioning
confidence: 99%
“…However, since this only affects a small portion of the quantified proteins, in fact those that are regulated, its global impact on the quantification accuracy is rather small. Nonetheless, recently developed multi--notch approaches [38,41], which are fully compatible with our workflow, but require special MS instrumentation that was not available for this study, have shown to reduce ratio distortion and should further increase quantification accuracy of our approach. We next tested if these calculated MS intensities can also be employed for estimating absolute protein concentration within the single samples using the recently introduced iBAQ approach [14,25,38].…”
Section: System--wide Estimation Of Protein Abundances In S Pombementioning
confidence: 99%