| Autor: |
Modzel M; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark.; Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland., Wollenberg DTW; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark.; Novozymes A/S, Krogshøjvej 36, Bagsværd 2880, Denmark., Trelle MB; Department of Clinical Biochemistry, Svendborg Hospital, Baagøes Allé 15, SVB Building 17.01, 5700 Svendborg, Denmark., Larsen MR; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark., Jørgensen TJD; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark. |
| Abstrakt: |
Ultraviolet photodissociation (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y, and c/z) are typically observed. Here we provide a detailed investigation of the level of H/D scrambling for all fragment ion types upon UVPD of the peptide scrambling probe P1 (HHHHHHIIKIIK) using an Orbitrap tribrid mass spectrometer equipped with a solid-state 213 nm UV laser. The most abundant UVPD-generated fragment ions (i.e., b/y ions) exhibit extensive H/D scrambling. Similarly, a/x and c/z ions have also undergone H/D scrambling due to UV-induced heating of the precursor ion population. Therefore, dominant b/y ions upon UVPD of protonated peptides are a strong indicator for the occurrence of extensive H/D scrambling of the precursor ion population. In contrast to peptide P1, UV-irradiation of ubiquitin did not induce H/D scrambling in the nonfragmented precursor ion population. However, the UVPD-generated b 2 and a 4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended. |
