Accelerating photofragmentation UV Spectroscopy-Mass spectrometry fingerprinting for quantification of isomeric peptides

Identification of isomeric biomolecules remains a challenging analytical problem. A recently developed spectroscopic method that combines UV photofragmentation and mass spectrometry for fingerprinting of cold ions (2D UV-MS), has already demonstrated its high performance in the library-based identification and quantification of different types of biomolecular isomers. The practical use of the method has been hindered by a slow rate of data acquisition, which makes the fingerprinting incompatible with high-throughput analysis and online liquid chromatography (LC) separation. Herein we demonstrate how the use of a few pre-selected wavelengths can accelerate the method by two orders of magnitude without a significant loss of accuracy. As a proof of principle, 2D UV-MS fingerprinting was coupled to online LC separation and tested for quantification of isomeric peptides containing either Asp or isoAsp residues. The relative concentrations of the peptides mixed in solution have been determined, on average, with better than 4% and 6% accuracy for resolving and non-resolving gradients of LC separation, respectively.

Automated summary

A spectroscopic method combining UV photofragmentation and mass spectrometry has been developed for identification and quantification of isomeric biomolecules, with the use of pre-selected wavelengths to accelerate data acquisition and maintain high accuracy. The method has been successfully applied to quantify isomeric peptides and achieved high accuracy under different LC separation gradients.