Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling

Abstract

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 that 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 is 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 non-fragmented precursor ion population. However, the UVPD-generated b2 and a4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…An additional strength of UVPD is that it can be applied for fragmentation of singly charged peptides, which cannot be examined by ETD/ECD techniques. Systematic examination of UVPD parameters revealed long UV irradiation can induce high levels of scrambling, even before fragmentation occurs . Only below 25 ms UV irradiation was there a low level of scrambling, which is consistent with previous uses of UVPD for HDX-MS .…”

“…Systematic examination of UVPD parameters revealed long UV irradiation can induce high levels of scrambling, even before fragmentation occurs. 259 Only below 25 ms UV irradiation was there a low level of scrambling, which is consistent with previous uses of UVPD for HDX-MS. 251 The study highlights the need to keep the irradiation times short to obtain fragments for accurately measuring deuterium content. The increased availability of instrumentation capable of electron- or photon-based dissociation, and likely additional novel approaches for peptide fragmentation in the future, is expected to propel the use of site-specific HDX-MS studies in the coming years.…”

Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology

“…Thus, the HDX rate of backbone amides is a structureand dynamics-sensitive probe. To get submolecular structure/ dynamics information by MS, one can use bottom-up workflows (which involve acid quenching of the HDX reaction followed by proteolysis using an acid protease and analysis of the resulting peptides), 126 top-down workflows (which involve ionization of the intact, labeled protein followed usually by electron-capture dissociation (ECD)-based fragmentation, 127 although electrontransfer dissociation (ETD) 128 and UV photodissociation 129 have also been used), or middle-down workflows (which combine limited proteolysis with nonergodic fragmentation). 130 Of these workflows, bottom-up generally provides the lowest structural resolution, associated with the length of the peptides generated during protease digestion; however, it is also the most versatile approach and is therefore by far the most widely used.…”

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides

“…It is a slow heating method, similar to CAD, and the energy is distributed throughout the peptide, resulting in bond cleavage at the weakest points (b and y fragments, labile posttranslational modifications). , Infrared (IR) radiation is resonant with the phosphate vibrational modes of the phosphorylated peptide, , leading to enhanced fragmentation of phosphopeptides. − Ultraviolet photodissociation (UVPD) at 193 nm deposits the required energy for dissociation within the absorption of a single photon (6.4 eV per photon) emitted through a nanosecond-scale laser pulse . The peptide absorbs the high-energy photon at the chromophore and the protein backbone amide group, accessing many dissociation pathways, leading to the generation of a/x, b/y, and c/z complementary ion pairs. , The fast fragmentation of the peptide reduces the frequency of phosphate group loss …”

“…49 The peptide absorbs the highenergy photon at the chromophore and the protein backbone amide group, 50 accessing many dissociation pathways, leading to the generation of a/x, b/y, and c/z complementary ion pairs. 51,52 The fast fragmentation of the peptide reduces the frequency of phosphate group loss. 53 The 12 T solariX FT-ICR mass spectrometer is a versatile instrument.…”