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mProphet: automated data processing and statistical validation for large-scale SRM experiments

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Selected reaction monitoring (SRM) is a targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected proteins at high sensitivity, reproducibility and accuracy. Currently, data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria, precluding the consistent analysis of different data sets and an objective assessment of their error rates. Here we present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.

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Figure 1: Structure of SRM data and definition of terms.
Figure 2: Generation of a gold-standard data set with assigned true peak groups.
Figure 3: Combining features improves the separation of true and false peak groups.
Figure 4: Separation of true from false peak group signals in a total human u2os cell line lysate using decoy transitions and mProphet scoring.

Change history

  • 06 April 2011

    In the version of this article initially published online, a 'greater than' sign was inadvertently reversed, and an author contribution was incorrectly attributed. The error has been corrected for the print, PDF and HTML versions of this article.


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We thank J. Malmström and M. Jovanovic for providing the samples that were used as background matrix in the gold-standard data set, M. Jovanovic for careful reading of the manuscript, A. Srebniak for help in generating a software package, and H. Wenschuh. We acknowledge M. Claassen for discussions on machine learning. This work was supported by grants from the Forschungskredit of the University of Zurich, University of Zurich Research Priority Program in Systems Biology and Functional Genomics, GEBERT-RÜF Stiftung and Swiss National Science Foundation (grant 31000-10767), with funds from the US National Heart, Lung, and Blood Institute and the US National Institutes of Health (contract N01-HV-28179), and by, the Swiss initiative for systems biology.

Author information

Authors and Affiliations



L.R., O.R., P.P., M.-Y.B. and R.A. designed the gold-standard data set. P.P. carried out the measurements on the gold-standard data set. L.R., O.R. and R.A. wrote the paper. L.R. and O.R. wrote the software and did the data analysis. L.R. did most of the statistical data analysis. R.H. contributed to the experiment involving the human plasma N-glycopeptide-enriched samples. M.B. contributed to the experiment involving the human u2os cell line. M.O.H. provided critical input on the project. R.A. supervised the project.

Corresponding author

Correspondence to Ruedi Aebersold.

Ethics declarations

Competing interests

O.R. and L.R. are employees of Biognosys AG. This company funded parts of the work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Table 1, Supplementary Results and Supplementary Note (PDF 7030 kb)

Supplementary Data 1

Table of transitions, table of peak groups, table with identification statistics and classifier of the gold standard data set analysis. The transitions sheet contains the precursor m/z (Q1), fragment ion m/z (Q3), an id that groups the transitions according to precursor (transition group id), an id for the transition (transition id), a string describing the isotopic labeling of the peptide (isotype), the collision energy used (CE), the expected retention time used for scheduled SRM (tR), the expected relative intensity of the fragment ions (relative intensity %), a string indicating whether the transition is a decoy or target (decoy) and an id to group corresponding target and decoy transition groups (target decoy transition group id). The mProphet peak groups sheet contains a row for each peak group. The most important columns are an id for a transition group measurement (transition_group_record), the features used for scoring (all columns starting with main_var or var_), a column indicating the dilution of the synthetic peptides in the specific matrix (dilution), the species used for the background matrix (background), the class of the peak group in terms of identity as determined by the dilution alignment (real_class), a boolean indicating whether the peak group was derived from decoy or target transitions (real_decoy), a boolean indicating whether treated as decoy or target in the mProphet analysis (decoy) and the mProphet discrimination score (d_score). The mProphet all peak groups sheet contains the all peak groups of the analysis, not only the ones that rank highest in one transition group record (peak_group_rank). The mProphet stat sheet relates the mProphet discrimination score (cutoff) to the false discovery rate (FDR) and the sensitivity (sens). The mProphet classifier weight sheet contains the weights that were determined using the semi-supervised learning approach. (XLS 2515 kb)

Supplementary Data 2

Table of transitions, table of peak groups, table with identification statistics and classifier of the human u2os cell line analysis. For a detailed description of the sheets see Supplementary Data 1 legend. (XLS 3791 kb)

Supplementary Data 3

Table of transitions, table of peak groups, table with identification statistics and classifier of the human plasma analysis. For a detailed description of the sheets see Supplementary Data 1 legend. (XLS 1166 kb)

Supplementary Data 4

Table of transitions and peak groups for the measurement of yeast target and decoy transitions in human plasma. The transitions sheet contains target transitions of yeast peptides and corresponding decoy transitions generated by two different decoy transition generation algorithms (ADD_RANDOM and REVERSE_PEP_AND_INCREASE_Q1). The mQuest peak groups sheet contains the data processed with mQuest. The mProphet analysis does result in meaningful results since the data contains no positive target measurements. For a detailed description of the sheets see Supplementary Data 1 legend. (XLS 675 kb)

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Reiter, L., Rinner, O., Picotti, P. et al. mProphet: automated data processing and statistical validation for large-scale SRM experiments. Nat Methods 8, 430–435 (2011).

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