Using ProtMAX to create high-mass-accuracy precursor alignments from label-free quantitative mass spectrometry data generated in shotgun proteomics experiments

Journal name:
Nature Protocols
Volume:
8,
Pages:
595–601
Year published:
DOI:
doi:10.1038/nprot.2013.013
Published online

Abstract

Recently, new software tools have been developed for improved protein quantification using mass spectrometry (MS) data. However, there are still limitations especially in high-sample-throughput quantification methods, and most of these relate to extensive computational calculations. The mass accuracy precursor alignment (MAPA) strategy has been shown to be a robust method for relative protein quantification. Its major advantages are high resolution, sensitivity and sample throughput. Its accuracy is data dependent and thus best suited for precursor mass-to-charge precision of ∼1 p.p.m. This protocol describes how to use a software tool (ProtMAX) that allows for the automated alignment of precursors from up to several hundred MS runs within minutes without computational restrictions. It comprises features for 'ion intensity count' and 'target search' of a distinct set of peptides. This procedure also includes the recommended MS settings for complex quantitative MAPA analysis using ProtMAX (http://www.univie.ac.at/mosys/software.html).

At a glance

Figures

  1. Workflow diagram from MS analysis to a quantitative protein data matrix.
    Figure 1: Workflow diagram from MS analysis to a quantitative protein data matrix.
  2. Target list example.
    Figure 2: Target list example.

    This file is a tab-delimited text file with two columns. Column A contains m/z values; column B contains Rt in minutes.

  3. ProtMAX output file.
    Figure 3: ProtMAX output file.

    Samples 1 and 2 correspond to the test sample in the Equipment section. (a) Charge state (no information for target approach). (b) Sum across all samples. (c) Sum of ion intensity counts specific for sample 1 (can also be SC or Ion count, depending on the selected method). (d) Scan number of the most intense ion signal of the corresponding m/z value specific for sample 1. (e) Rt of the most intense ion signal of the corresponding m/z value specific for sample 1.

  4. Graphical user interface of the Xcalibur MS instrument setup.
    Figure 4: Graphical user interface of the Xcalibur MS instrument setup.

    The settings for the protein analysis are indicated by green circles that are numbered according to the steps in Box 1 (Instrument settings for the protein shotgun LC-MS/MS analysis using an Orbitrap) in which they are described. (a) MS1 settings and internal Lock Mass calibration. (b) MS/MS Scan Events. (c) Data-dependent settings for MS/MS scan events.

  5. Graphical user interface of ProtMAX.
    Figure 5: Graphical user interface of ProtMAX.

    The settings for the unbiased peptide matrix generation are indicated by green circles that are numbered according to the PROCEDURE steps in which they are described.

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Author information

Affiliations

  1. Department of Molecular Systems Biology, University of Vienna, Vienna, Austria.

    • Volker Egelhofer,
    • Wolfgang Hoehenwarter,
    • David Lyon,
    • Wolfram Weckwerth &
    • Stefanie Wienkoop
  2. Present address: Proteome Analysis Research Group, Leibniz Institute for Plant Biochemistry, Halle, Germany.

    • Wolfgang Hoehenwarter

Contributions

V.E. developed the algorithm of the ProtMAX tool. W.H. contributed to the concept and optimization of the ProtMAX tool of protein analysis and writing of the manuscript. D.L. contributed to the concept of the ProtMAX tool and writing of the manuscript. W.W. conceived the concept of the MAPA strategy for proteomics. S.W. conceived Preferences and feature upgrading with the target approach of the ProtMAX tool and was responsible for project coordination and writing of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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