Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry

Abstract

Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.

Direct Ultraviolet Laser-Induced Reduction of Disulfide Bonds in Insulin and Vasopressin

“…A trisulfide bond was also observed in vasopressin at neutral pH. 28 Overall, these data suggest that UV light-induced disulfide scrambling may lead to trisulfide bonds in proteins. Interestingly, trisulfide bond impurities have previously been reported in recombinant monoclonal antibodies.…”

“…This product was identified as dithiohemiacetal-vasopressin, previously also identified as scrambled dimeric species at neutral pH as well as observed in the vasopressin homolog oxytocin. 28,31 The disulfide bond in somatostatin-14 was stable toward photoconversion despite the presence of a Trp residue. No photoproduct involving disulfide bond reduction was identified at acidic pH, consistent with the previous study on the peptide at neutral pH where no intermolecular disulfide scrambling was detected.…”

“…No photoproduct involving disulfide bond reduction was identified at acidic pH, consistent with the previous study on the peptide at neutral pH where no intermolecular disulfide scrambling was detected. 28 The photoreduction yield of insulin B-chain and vasopressin was 2−3%. The quantities were enough for LC-MS/MS (-MS 2 ) analysis after 60 s of laser irradiation, and the fragmentation spectra of the photoreduced products provided improved sequencing due to the removal of the disulfide bonds, supporting that the products were reduced ( Figure S1).…”

“…The M−NH 3 Tyr photo-modification was previously identified for vasopressin at neutral pH and contained a new local maximum of 294 nm instead of the classical Tyr profile. 28 Characterization of the chemically reduced deaminated product revealed that the mass loss was localized in the a 2 /b 2 (Cys1−Tyr2−) pair fragments ( Figure S3). Also, the Tyr immonium ion was absent in the MS 2 fragmentation.…”

“…32,37 Besides the novel Phe−Trp cross-link in somatostatin-14, the conversion of Trp to the common photooxidation product N-formylkynurenine was also observed, consistent with UV laser stress at neutral pH (data not shown). 28 NFK-somatostatin-14 was for the first time reported as a product from fluorescent light. 38 Established Tyr oxidations were searched for in arginine vasopressin and human insulin.…”