Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale
Intact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identiﬁcation and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in conﬁdent identiﬁcation of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identiﬁed in both the UVPD and optimized HCD analyses demonstrated a substantial increase in conﬁdence in identiﬁcation (as deﬁned by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)−UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identiﬁed and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modiﬁcation.