Quantitation: Replicate protocol
The replicate protocol is used for label-free quantitation based on the relative intensities of extracted ion chromatograms (XICs) for precursors in multiple data sets aligned using mass and elution time.
This protocol requires information from the raw data file that is not present in the peak list, so the quantitation report is generated in Mascot Distiller, which has access to both the Mascot search results and the raw data.
Literature
The accuracy of this protocol is highly dependent on good mass accuracy and reproducible chromatography, so it is not surprising that instrument manufacturers have played a major part in its development. In no particular order, a few key publications are:
- Wang, W. X., et al., Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards, Analytical Chemistry 75 4818-4826 (2003)
- Chelius, D., et al., Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry, Analytical Chemistry 75 6658-6665 (2003)
- Radulovic, D., et al., Informatics platform for global proteomic profiling and biomarker discovery using liquid chromatography-tandem mass spectrometry, Molecular & Cellular Proteomics 3 984-997 (2004)
- Silva, J. C., et al., Quantitative proteomic analysis by accurate mass retention time pairs, Analytical Chemistry 77 2187-2200 (2005)
- Hughes, M. A., et al., Quantitative proteomic analysis of drug-induced changes in mycobacteria, Journal of Proteome Research 5 54-63 (2006)
Configuration tips
- Your Mascot Server must be 2.2.06 or later. An updated copy of quantitation.xml is available for Mascot Server version 2.2 (only). Right click the link and choose Save As.
- If you wish to use another application to identify features that are up or down regulated, and generate an inclusion list for a mass spectrometer to obtain MS/MS data for these features, you can then build a Distiller project that includes the set of MS files and the single MS/MS file. Just omit the MS/MS file from the ratios to be reported. Distiller will happily use the MS/MS to locate the set of features to be quantified across all the MS files.
- Always check Allow elution shift and ensure the maximum shift is set to an appropriate value.
Examples
To see how this method works in practice, try Distiller on 30 day evaluation. Follow through the tutorial in the Distiller help (F1) to become familiar with the way Distiller works, then process your own data. If you don’t have an in-house Mascot Server, you can use the free, public Mascot server, as long as you stay within the limit on the number of spectra. If you have an in-house server, no such limits apply, but make sure it is patched to 2.2.06 or later.
