Label free quantitation

Label free quantitation, also referred to as no label quantitation, is based on precursor signal intensity, which is, in most cases applied to data acquired on high mass precision spectrometers equipped with the new generation of time-of-flight (Tof), Fourier transform-ion cyclotron resonance (FTLTQ), or OrbiTrap mass analyzers. The high resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process.

The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides.

Detecting Peptides

Typically, peptide signals are detected at the MS1 level and distinguished from chemical noise through their characteristic isotopic pattern. These patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the mono-isotopic peptide mass. The total ion current of the peptide signal is then integrated and used as a quantitative measurement of the original peptide concentration. For each detected peptide, all isotopic peaks are first found and the charge state is then assigned.

Matching Corresponding Peptides

In contrast to differential labelling, every biological specimen needs to be measured separately in a label-free experiment. The extracted peptide signals are then mapped across few or multiple LC-MS measurements using their coordinates on the mass to charge and retention time dimension. Data from high mass precision instruments greatly facilitate this process and increase the certainty of matching correct peptide signals across runs. In addition to the m/z dimension, the TR coordinate is used to map corresponding peptides between runs.

electing Discriminatory Peptides

Finally, sophisticated normalization methods are used to remove systematic artefacts in the peptide intensity values between LC-MS measurements. Then, discriminatory peptides are identified by selecting the peptides whose normalized intensities are different (e.g., p-value < 0.05) among multiple groups of samples.

In addition, newer hybrid mass spectrometers like LTQ OrbiTrap offer the possibility to acquire MS/MS peptide identifications in parallel to the high mass precision measurement of peptides on the MS1 level. This raises the computational challenge for the processing and integration of these two sources of information and has led to the development of novel promising quantification strategies.

References

*cite journal
author=Lukas N. Mueller et al.| date=2008
journal=Journal of Proteome Research
volume=7
pages=51–61
doi=10.1021/pr700758r
title=An Assessment of Software Solutions for the Analysis of Mass Spectrometry Based Quantitative Proteomics Data