How Much Do I Have?
As researchers press further into the understanding of life sciences, quantification provides greater insight into proteomic mysteries.
Correct peak picking, also known as feature detection and discarding noise peaks from any samples are vitally important. Using highly tuned algorithms, PEAKS Q delivers confidence through increased result accuracy and improved statistics.
The quantification application of PEAKS works seamlessly across all current experimental approaches:
- ICAT (Isotope Coded Affinity Tags)
- ICPL (Isotope Coded Protein Labels)
- iTRAQ (Isobaric Tag for Relative and Absolute Quantification)
- Label Free Quantification
- N-Terminal Labeling (e.g. methylation)
- O18
- SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture)
- TMT (Tandem Mass Tags)
- User Defined Labeling
The goal is to calculate the relative abundance of proteins. To do so, PEAKS Q uses a set of peptides identified as matching to a protein. In this way, protein quantification relies on protein identification; PEAKS database search engine and de novo sequencing are highly recommended methods. Calculating the abundance ratios between samples for each peptide is easily achieved. Further, flexible normalization functions are available to compare various data sets.
When accounting for differential modification, it should be noted that not all peptide ratios from a given protein are the same. It is the job of the statistic package to decide which peptide ratios to trust and which to discard as outliers. The current PEAKS Q uses Dixon's Q-test for this purpose.
Even after removing outliers, some peptide ratios should be trusted over others. For instance, one ratio might be calculated from a very clean spectrum that has been matched to a peptide ID with high confidence; another might be from a noisy spectrum. The analysis should consider these factors and give a trustworthy score or weight, to each ratio. The protein ratio is the weighted average of the peptide ratios, normalized protein ratios.
For more information about labeled or label free techniques, click on the corresponding links:
Visual Inspection
Visual inspection of raw spectra becomes very important. It allows researchers to adjust the parameter setting to gain much more accurate ratios. The PEAKS quantification package provides a 2D view of the MS/MS spectrum as well as a 3D view of the parent scan for each identified peptide. Thus the actual ratios can be easily verified by inspecting the correspondent raw spectra.
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