Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 2;8(11):933-5.
doi: 10.1038/nmeth.1716.

Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging

Craig D Wenger  1 , M Violet Lee, Alexander S Hebert, Graeme C McAlister, Douglas H Phanstiel, Michael S Westphall, Joshua J Coon
Affiliations

Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging

Craig D Wenger et al. Nat Methods. .

Abstract

We describe a mass spectrometry method, QuantMode, which improves accuracy of isobaric tag-based quantification by alleviating the pervasive problem of precursor interference, simultaneous isolation and fragmentation of impurities, through gas-phase purification. QuantMode analysis of a yeast sample 'contaminated' with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan, with a small loss of spectral identifications. This technique enables large-scale, multiplexed quantitative proteomics using isobaric tagging.

PubMed Disclaimer

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare competing financial interests: Two patent applications, in part related to this manuscript, are pending. US Serial Numbers 13/086638 (inventors Wenger CD, Phanstiel DP, and Coon JJ) and 61/471461 (inventors Coon JJ and Westphall MS).

Figures

Figure 1
Figure 1
Analysis of the precursor purity model and quantitative accuracy model samples with either HCD MS/MS or QuantMode. (a) Distribution of precursor purity as measured by examining reporter tag 126 (yeast) and 131 (human) for yeast-identified sequences using either HCD MS/MS (red) or QuantMode(blue). (b) Analysis of quantitative accuracy via HCD MS/MS (left), HCD MS/MS with PAF (middle), and QuantMode (right). The dashed red horizontal line indicates the true ratio while boxplots indicate the median (stripe), the 25th to 75th percentile (interquartile range, box), 1.5 times the interquartile range (whiskers), and outliers (open circles). The number of quantified yeast PSMs (N) and median ratio are given for each method.
Figure 2
Figure 2
Pedagogical overview of QuantMode. A triply charged precursor at m/z 595.72 was isolated with a 3 Th window (a). The precursor isotopic cluster occupies only 49% of the total ion current in this region. QuantMode begins with PTR (b); isolation of the charge-reduced precursor (+2) purifies this target to 85% (c); HCD converts these purified precursors to reporter ions; resonant-excitation CAD follows re-injection/re-isolation of the triply charged precursor. The HCD and CAD products are combined in the c-trap prior to orbitrap mass analysis. The conventional HCD MS/MS scan for this impure precursor (d) is juxtaposed against this QuantMode scan (e). Insets display the reporter ion region (identical intensity scale) and the quantitative accuracy achieved by both approaches for the 10:1 ratio.
See this image and copyright information in PMC

Comment in

References

    1. de Godoy LMF, et al. Nature. 2008;455:1251–1255. - PubMed
    1. Ong SE, Mann M. Nat Chem Biol. 2005;1:252–262. - PubMed
    1. Jiang H, English AM. J Proteome Res. 2002;1:345–350. - PubMed
    1. Ong SE, et al. Mol Cell Proteomics. 2002;1:376–386. - PubMed
    1. Olsen JV, et al. Sci Signal. 2010;3:ra3. - PubMed

Publication types