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Characterization of Unbranched Ubiquitin Tetramers by Combining Ultraviolet Photodissociation with Proton Transfer Charge Reduction Reactions

ABSTRACT: Characterization of six unbranched ubiquitin tetramers linked at various Lys using UVPD followed by PTCR

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jennifer Brodbelt

PROVIDER: PXD043027 | JPOST Repository | Sun Jun 16 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	050923_K11Ub4_IW=2_25+_0.8mJ_2iso1139w60_PTR10.raw	Raw
	050923_K11Ub4_IW=2_25+_0.8mJ_2iso1189w60_PTR10.raw	Raw
	050923_K11Ub4_IW=2_25+_0.8mJ_2iso1239w60_PTR10.raw	Raw
	050923_K11Ub4_IW=2_25+_0.8mJ_2iso1289w60_PTR10.raw	Raw
	050923_K11Ub4_IW=2_25+_0.8mJ_2iso1339w60_PTR10.raw	Raw

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Ultraviolet Photodissociation of tryptic peptide backbones at 213 nm

Project description:We analyzed the backbone fragmentation behavior of tryptic peptides of a four protein mixture and of E. coli lysate subjected to Ultraviolet Photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15,178 high-confidence peptide-spectrum matches by additionally recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor. Type a, b and y ions were most prominent in UVPD spectra and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity) and remaining precursor intensity high. Sequence coverage and sequence fragment intensity ratio correlated with precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to non-covalent interactions. UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points at aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange and on spectrumviewer.org/db/UVPD_213nm_trypPep

2020-05-07 | PXD018176 | Pride

Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization.

Project description:Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C-Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C-N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization. Graphical Abstract ᅟ.

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Ultraviolet Photodissociation of ESI and MALDI generated protein ions on a Q-Exactive mass spectrometer

Project description:UVPD was implemented on an Orbitrap Q-Exactive plus equipped with a ESI/EP-MALDI. UVPD of MALDI generated ions was benchmarked against MALDI-ISD, MALDI-HCD and ESI-UVPD. MALDI UVPD outperformed MALDI HCD and ISD efficiently sequencing small proteins ions.

2019-01-24 | PXD011526 | Pride

Structural Characterization of Glycosaminoglycan Carbohydrates Using Ultraviolet Photodissociation.

Project description:Structural characterization of sulfated glycosaminoglycans (GAGs) by mass spectrometry has long been a formidable analytical challenge owing to their high structural variability and the propensity for sulfate decomposition upon activation with low-energy ion activation methods. While derivatization and complexation workflows have aimed to generate informative spectra using low-energy ion activation methods, alternative ion activation methods present the opportunity to obtain informative spectra from native GAG structures. Both electron- and photon-based activation methods, including electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), and extreme ultraviolet photon activation, have been explored previously to overcome the limitations associated with low-energy activation methods for GAGs and other sulfated oligosaccharides. Further, implementation of such methods on high-resolution mass spectrometers has aided the interpretation of the complex spectra generated. Here, we explore ultraviolet photodissociation (UVPD) implemented on an Orbitrap mass spectrometer as another option for structural characterization of GAGs. UVPD spectra for both dermatan and heparan sulfate structures display extensive fragmentation including both glycosidic and cross-ring cleavages with the extent of sulfate retention comparable to that observed by EDD and NETD. In addition, the relatively short activation time of UVPD makes it promising for higher throughput analysis of GAGs in complex mixtures.

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Characterization of disulfide linkages in proteins by 193 nm ultraviolet photodissociation (UVPD)

Project description:Inter-linked disulfide bonds connecting peptide chains are homolytically cleaved with 193 nm ultraviolet photodissociation (UVPD). Analysis of insulin demonstrates the ability for UVPD to cleave multiple disulfide bonds and provide sequence coverage of multiple peptide chains in the same MS/MS event. The information provided by UVPD of disulfide-bound peptides is comparable to information garnered from other high energy dissociation methods but requires an activation period an order of magnitude faster than these methods. This phenomenon was investigated, along with a partial reduction and alkylation sample preparation to determine disulfide connectivities of enzymatically digested proteins. The 4 disulfide bonds of lysozyme and the 19 disulfide bonds of serotransferrin were unambiguously characterized through non-reduced and partially reduced protein digestions.

2018-04-28 | PXD009460 | JPOST Repository

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Project description:Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein-ligand and protein-protein complexes and to provide auxiliary information about the binding sites of the ligands and protein-protein interfaces. UVPD outperformed collisional induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) in terms of yielding the most comprehensive diagnostic primary sequence information about the proteins in the complexes. UVPD also generated noncovalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m(7)GTP, and human peptidyl-prolyl cis-trans isomerase 1 (Pin1) in complex with the peptide derived from the C-terminal domain of RNA polymerase II (CTD). Noncovalently bound protein-protein fragment ions from oligomeric ?-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.

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Project description:Advances in mass spectrometry have made it a preferred tool for structural characterization of glycerophospholipids. Collisional activation methods commonly implemented on commercial instruments do not provide fragmentation patterns that allow elucidation of certain structural features, including acyl chain positions on the glycerol backbone and double bond positions within acyl chains. In the present work, 193 nm ultraviolet photodissociation (UVPD) implemented on an Orbitrap mass spectrometer is used to localize double bond positions within phosphatidylcholine (PC) acyl chains. Cleavage of the carbon-carbon bonds adjacent to the double bond provides a diagnostic mass difference of 24 Da and enables differentiation of double-bond positional isomers. The UVPD method was extended to the characterization of PCs in a bovine liver extract via a shotgun strategy. Positive mode higher energy collisional dissociation (HCD) and UVPD, and negative mode HCD were undertaken in a complementary manner to identify species as PCs and to localize double bonds, respectively.

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Project description:We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified c- and z-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS(3) format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.

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Enhanced Characterization of Membrane Protein Complexes by Ultraviolet Photodissociation Mass Spectrometry.

Project description:Development of chemical chaperones to solubilize membrane protein complexes in aqueous solutions has allowed for gas-phase analysis of their native-like assemblies, including rapid evaluation of stability and interacting partners. Characterization of protein primary sequence, however, has thus far been limited. Ultraviolet photodissociation (UVPD) generates a multitude of sequence ions for the E. coli ammonia channel (AmtB), provides improved localization of a possible post-translational modification of aquaporin Z (AqpZ), and surpasses previous reports of sequence coverage for mechanosensitive channel of large conductance (MscL). Variations in UVPD sequence ion abundance have been shown to correspond to structural changes induced upon some perturbation. Preliminary results are reported here for elucidating increased rigidity or flexibility of MscL when bound to various phospholipids.

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