For most researchers the goal of an LC-MS proteomics experiment is a list of proteins showing significant expression differences. In most cases what you actually measure, whether you use a labelled or label-free approach, are signals from peptide ions for the protein of interest. The challenge is how to recombine all these individual peptide ion measurements and make sense of what that means at the protein level. There are several ways to achieve this but Progenesis LC-MS gives a reliable measure of relative protein abundance for label-free experiments because of the unique way it works.
First, all the runs are aligned and an aggregate image is used to produce a feature map. This common set of features is applied to all individual runs in your experiment, so the software quantifies the same peptide ions across all the aligned runs. Peptide ions with fragmentation spectra are searched against relevant databases. Any identifications that are returned, which meet your search criteria, are automatically linked to the same feature in every run. To measure protein abundance Progenesis LC-MS sums all the unique peptide ion abundances identified as coming from a specific protein.
Because you have exactly the same number of quantified peptides identified on all runs you can directly compare the abundance of a protein between groups. The software uses the summed peptide ion abundances to generate relative expression measure (fold-change) and the significance of any measured differences (ANOVA p-value).
The image above illustrates how this works for an example, “Protein X”, but you can read more in our technical note including examples of published data validating our approach.
Technical Note – Protein Quantification by Progenesis LC-MS
Benefits of this approach:
- Co-detection of features provides ion abundance measures for the same peptides from each protein on every run, with no missing values
- Summing means that ion abundances with higher signal, and therefore less “noisy”, have more weight; making the final protein quantification more reliable
- When two protein identifications share common peptides the software uses unique peptides for quantification
- Searching peptides against databases can return multiple entries that are actually the same or related proteins, so the software automatically groups similar proteins into one quantification result
- Fractionated proteins are quantified in the same way but include all peptides from every fraction
But don’t take our word for it! We encourage you to download Progenesis LC-MS and try our approach to protein quantification on your own data today.