Quantitation: Precursor protocol

Quantitation based on the relative intensities of extracted ion chromatograms (XICs) for precursors within a single data set. This is by far the most widely used protocol, which can be used with any chemistry that creates a precursor mass shift. For example,

• ¹⁸O: Miyagi, M. and Rao, K. C. S., Proteolytic O-18-labeling strategies for quantitative proteomics, Mass Spectrometry Reviews 26 121-136 (2007)
• AQUA: Gerber, S. A., et al., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS, Proceedings of the National Academy of Sciences of the United States of America 100 6940-6945 (2003)
• ICAT: Gygi, S. P., et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nature Biotechnology 17 994-999 (1999)
• ICPL: Schmidt, A., et al., A novel strategy for quantitative proteomics using isotope-coded protein labels, Proteomics 5 4-15 (2005)
• Metabolic: Beynon, R. J. and Pratt, J. M., Metabolic labeling of proteins for proteomics, Molecular & Cellular Proteomics 4 857-872 (2005)
• SILAC: Ong, S. E., et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Molecular & Cellular Proteomics 1 376-386 (2002)

The precursor 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.

Ideally, the survey scan raw data should be true profile data with good mass resolution. For standard trap data, it can be difficult to get good quantitation off survey scans, and zoom scans will give better results.

Where to begin

You need Mascot Distiller, including the Search Toolbox and Quantitation Toolbox, and access to a Mascot Server. You can automate all stages of processing using Mascot Daemon, except for multi-file projects. The Distiller online help contains detailed descriptions of how to proceed.

An example workflow is shown in Diethylation, Dimethylation and the Deuterium effect.

Configuration tips

• An updated copy of quantitation.xml is available for Mascot Server 2.2 (only). Right click the link and choose Save As.
• It is not unusual to get MS/MS for just one component of a pair. Check Allow mass time match (allow_mass_time_match, required) to enable the partner to be inferred from its predicted mass. If allow_mass_time_match is false, then all_charge_states will be treated as false, even if true in the method.
• Do not check Allow elution shift unless your tag includes Deuterium, which causes a systematic shift in elution time. With other tags, setting this option to true slows down processing and may degrade the results for weak signals that give noisy XIC’s.

Examples

To see screen shots of examples of Precursor quantitation, Diethylation, Dimethylation and the Deuterium effect.

A better way to see how it works is to try Distiller on 30 day evaluation and follow through the tutorial in the help file. This uses data from a three component SILAC experiment acquired on an AB SCIEX QStar instrument. You can follow the tutorial and experiment with the processing settings even if you don’t have an in-house Mascot Server.