Label-Free Quantitation

In a LFQ DDA approach, the ions for a given m/z range are individually isolated and fragmented. The quantitation involves extracting peptide chromatograms (MS ¹ precursor ion) from LC-MS runs and integrating peak areas over the chromatographic time scale or using the intensity at the highest point of the chromatographic peak. The resulting areas or intensities are compared across sample set (i.e. control vs experimental) for quantitation.

In traditional data-dependent workflows, consistent precursor and thereby protein quantitation can be challenging to achieve due to the stochastic sampling nature of the mass spectrometer. Innovations in software algorithms now extract LC-MS peaks in the raw data files and map them to identified spectra, detecting these features across LC-MS data files using retention-time alignment and feature linking, minimizing "missing data points" and maximizing quantitative insights.

In a LFQ DIA approach, a precursor mass range is selected and then divided into a series of relatively wide isolation windows: for example, 25 m/z each. Chimeric MS/MS data is acquired from all detected precursor ions in the first isolation window and then repeated for each consecutive, adjacent isolation window until the entire precursor mass range is covered. MS/MS spectral libraries are used to identify peptides of interest from the acquired data.

DIA significantly increases coverage and reproducibility by acquiring MS/MS data from all detected precursor ions; it also makes retrospective data analysis possible. LFQ DIA involves extracting peptide chromatograms (MS ¹ precursor ion or MS ² fragment ions) from LC-MS runs and integrating peak areas over the chromatographic time scale, and resulting areas are compared across the sample set for quantitation. The samples that are part of the comparison study are run individually.