Photo-SRM: laser photo-dissociation improves detection selectivity of selected reaction monitoring mode
Résumé
Selected Reaction Monitoring (SRM) carried out on triple quadrupole mass spectrometers coupled to liquid chromatography has been a reference method to develop quantitative analysis of small molecules in biological or environmental matrices for years and is currently emerging as a promising tool in clinical proteomics. However, sensitive assays in complex matrices are often hampered by the presence of co-eluted compounds that share redundant transitions with the target species. In the present work we document for the first time the potential interest of substituting classical gas-collision activation mode by laser photodissociation to track chromophore-derivatized target peptide absorbing at 532 nm diluted in a whole plasma trypsin hydrolysate. We anticipate that this technique coined photo-SRM, combined with chromophores endowed with reactivity towards generic chemical groups, might significantly improve the limit of quantification of classical SRM-based assays. The present presentation focused on the development of a new technique dedicated to quantification, called photo-SRM, where non-discriminating collision-activated dissociation mode has been replaced by a more specific photo-dissociation process governed by the absorbing properties of a targeted compound. The first example photo-SRM experiments has been developed in-house on a conventional triple quadrupole mass spectrometer. Proof of principle of SRM-based monitoring by laser-induced dissociation instead of classical collision activated mode was carried out with the chromophore-derivatized-Oxytocin as a model molecule diluted in whole plasma trypsin hydrolysate. This preliminary investigation clearly demonstrates that both the selectivity and detection level may markedly be improved during SRM monitoring by replacing the classical mode of activation by gas collision by a photon excitation providing a judicious overlap between the absorbing properties of the target molecule and the excitation wavelength of the laser beam. The lack of cross contamination within the transition channel between the target molecule and co-eluted endogenous interferences results here in a 50-fold improvement of the limit of quantification. The question rises now about how photo-SRM might be further optimized and extended to molecules that do not exhibit natural or specific light absorption. Novel aspect: First demonstration of Photo-SRM tool for analytical quantification