Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry

Abstract

A chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating structures of proteins and protein complexes is reported, as demonstrated for lysozyme and beta-lactoglobulin with and without bound ligands. The chemical probe, NN, incorporates a UV chromophore that endows peptides with high cross sections at 351 nm, a wavelength not absorbed by unmodified peptides. Thus, NN-modified peptides can readily be differentiated from nonmodified peptides in complex tryptic digests created upon proteolysis of proteins after their exposure to the NN chemical probe. The NN chemical probe also affords two diagnostic reporter ions detected upon UVPD of the NN-modified peptide that provides a facile method for the identification of NN peptides within complex mixtures. Quantitation of the modified and unmodified peptides allows estimation of the surface accessibilities of lysine residues based on their relative reactivities with the NN chemical probe.

Photodissociation mass spectrometry: new tools for characterization of biological molecules

“…Brodbelt et al reported an amine-reactive reagent (Figure 28) that incorporated a UV chromophore at 351 nm, thus allowing the ability to pinpoint and dissociate the diagnostic probe-modified peptides among complex mixtures of unmodified peptides. 61,180 …”

Ion Activation Methods for Peptides and Proteins

“…Successful UVPD of peptides has been demonstrated using a variety of lasers, including F 2 excimer laser (157 nm, 7.9 eV per photon), 86 ArF excimer laser (193 nm, 6.4 eV per photon), 87 a femtosecond titanium sapphire laser (800 nm, 1.5 eV per photon), 88 a Nd:YAG laser (266 nm, 4.7 eV per photon 89–91 or 355 nm, 3.5 eV per photon 92 ), and a XeF excimer laser (351 nm, 3.5 eV per photon). 93 (Photodissociation has also been implemented using visible photon wavelengths, such as 473 nm.) 94,95 For each wavelength, UVPD requires a suitable chromophore for absorption of photons.…”

Implementing visible 473 nm photodissociation in a Q-Exactive mass spectrometer: towards specific detection of cysteine-containing peptides

“…21 The same experimental set-up also proved to be efficient in streamlining the detection of peptides tagged with a sulfo-NHS amine reactive chromophore group, which probes the accessibility of lysine residues at the protein surface. 22 Specific photodissociation was also obtained for Tyr/His-containing peptides after derivatization with diazonium chromophore groups. 23 Detection specificity is also critical when tackling protein quantification in whole proteome by targeted mass spectrometry in selected reaction monitoring mode (SRM 25 Thus, photo-SRM showed similar or even improved detection performance compared to conventional CID-SRM for the analysis of a whole plasma hydrolysate.…”