Label-free quantitation, an extension to 2DB

Abstract

Determining the differential expression of proteins under different conditions is of major importance in proteomics. Since mass spectrometry-based proteomics is often used to quantify proteins, several labelling strategies have been developed. While these are generally more precise than label-free quantitation approaches, they imply specifically designed experiments which also require knowledge about peptides that are expected to be measured and need to be modified. We recently designed the 2DB database which aids storage, analysis, and publication of data from mass spectrometric experiments to identify proteins. This database can aid identifying peptides which can be used for quantitation. Here an extension to the database application, named MSMAG, is presented which allows for more detailed analysis of the distribution of peptides and their associated proteins over the fractions of an experiment. Furthermore, given several biological samples in the database, label-free quantitation can be performed. Thus, interesting proteins, which may warrant further investigation, can be identified en passant while performing high-throughput proteomics studies.

Existing bioinformatics tools for the quantitation of post-translational modifications

“…: the spectra that were successfully assigned a peptide sequence) among samples from different experimental conditions (Washburn et al 2001;Liu et al 2004;Gilchrist et al 2006). In addition the count can be weighted by the total ion current of the counted spectra which can improve its dynamic range (Asara et al 2008;Allmer 2010). Alternatively, it can be weighted by the reported score of the identification software (Allet et al 2004); although one study showed that identification scores may not be strongly correlated with protein abundance (Ong and Mann 2005).…”

“…Since spectral counting is rather trivial, the counts are often generated by non specialized software such as PeptideProphet (Keller et al 2002) and MSMAG (Allmer 2010), or by specialized software or scripts (Gao et al 2003;Old et al 2005;Ishihama et al 2005;Huttlin et al 2007). …”

Relative protein quantitation with post translational modifications in mass spectrometry based proteomics

“…A heat map for peptides is found under the tab Peptide Overview and Peptide Details contains the numerical information for the analysis and for each fraction separately. Options such as using peptide standards for normalization are not detailed in this work; they have been presented when MSMAG was first introduced [25]. …”

“…One method to perform label-free quantitation with liquid chromatography (LC) MS/MS data is to perform spectral counting [21][22][23]. In addition to spectral counting, the count can be weighted by the total ion current (TIC) [24,25] or the reported score of the identification software [26]. MSMAG is an extension to 2DB and can be used for labelfree quantitation essentially as a new feature in the analysis and presentation module while the underlying data model remains unchanged [25].…”

“…In addition to spectral counting, the count can be weighted by the total ion current (TIC) [24,25] or the reported score of the identification software [26]. MSMAG is an extension to 2DB and can be used for labelfree quantitation essentially as a new feature in the analysis and presentation module while the underlying data model remains unchanged [25]. Since PTMs are also modeled in the database, it was natural to extend the quantitation facility and allow for quantitation of labeled analytes.…”