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Abstract Introduction
Targeted proteomics by multiple or parallel reaction monitoring (MRM/PRM) is the current gold standard for absolute protein quantification in complex biological samples. Quantification of target proteins is achieved by spiking analyte matrices with heavy-labelled standards in known amounts. However, methods using targeted acquisition require extensive and time-consuming method establishment and the number of target proteins per run is limited.
Recently, data-independent acquisition (DIA) has been established as the new standard for discovery proteomics. Rapid scanning of moving acquisition windows allows for comprehensive analysis of complex biological samples. DIA also enables reproducible and precise protein quantification, due to less missing values compared to standard datadependent acquisition (DDA) approaches. In contrast to DDA mode, eluting peptides are fragmented several times, allowing for quantification on the MS2 level. Standard DIA approaches are restricted to inter-run relative quantification, but do not allow for intra-run absolute quantification.
Objective
We seek to enable absolute quantification of target proteins in DIA approaches by using heavy labeled quantification standards.
Methods
To assess the linearity of target protein quantification and overall analytical depth by DIA, 27 peptides of an artificial protein standard were spiked at increasing concentrations into a HeLa digest. Samples were measured in DIA mode and analyzed for the linearity of target protein signal. In a second experiment, we spiked both labeled and unlabeled artificial protein into a human urine sample to proof the feasibility of intra-run absolute protein quantification.
Results & Discussion
In HeLa digest, we observed a linearity of target peptide intensities over four orders of magnitude. In the same experiment, we identified 5,835 HeLa proteins. In human urine, we were able to precisely quantify the spiked target protein, while identifying 835 human proteins.
Conclusion
Our proof-of-concept study shows that the deep protein coverage of DIA methods can be combined with exact target protein quantification in the same LC-MS/MS run. This opens the potential to quantify a high number of target proteins in complex samples.
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= Discovery stage.
= Translation stage.
= Clinically available.
