Dustin D. Holden

Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimers disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.

Assessment of Metabolic Stability Using the Rainbow Trout (Oncorhynchus mykiss) Liver S9 Fraction

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species‐specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold‐water species. Protocols for other species (e.g., carp, a warm‐water species) can be developed based on these procedures as long as the specific considerations are taken into account. Curr. Protoc. Toxicol. 53:14.10.1‐14.10.28.

Hybridizing Ultraviolet Photodissociation with Electron Transfer Dissociation for Intact Protein Characterization

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 MS3 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.

Shotgun Analysis of Rough-Type Lipopolysaccharides Using Ultraviolet Photodissociation Mass Spectrometry

Detailed structural characterization of intact rough-type lipopolysaccharides (R-LPS) was accomplished using a multi-stage mass spectrometry (MS(3)) strategy consisting of collision-induced dissociation (CID) followed by 193 ultraviolet photodissociation (UVPD) implemented on an Orbitrap Fusion mass spectrometer. Complex mixtures of R-LPS from either Escherichia coli or Salmonella enterica were directly infused into the mass spectrometer using static source nanoelectrospray ionization (nanoESI). An initial CID event performed on an R-LPS precursor produced spectra with abundant ions corresponding to the lipid A and core oligosaccharide (OS) substructures. Comparison of CID spectra of R-LPS ions with varying lipid A and core OS structures verifies that lipid A and core OS ions are consistently produced in high abundance. The resulting lipid A and core OS ions were subsequently activated by CID, high-energy collision-induced dissociation (HCD), or UVPD. For both the lipid A and core OS substructures, HCD and UVPD produced highly informative complementary spectra, with UVPD of the core OS producing an extensive array of cross-ring cleavage fragments. Successful discernment of E. coli R-LPS structures with isomeric core structures confirmed the degree to which subtle structural differences could be determined using this method.

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures

The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent. A hierarchical decision-tree MS(n) method is used in conjunction with a hybrid activation technique, UVPDCID, to acquire characteristic fragmentation patterns of lipid A variants from a number of Gram-negative bacteria. Structural assignments are derived from integration of key features from three to five spectra and automated interpretation is achieved in minutes without the need for pre-existing information or candidate structures. The utility of this strategy is demonstrated for a mixture of lipid A structures from an enzymatically modified E. coli lipid A variant. A total of 27 lipid A structures were discovered, many of which were isomeric, showcasing the need for a rapid de novo approach to lipid A characterization.

Integration of Ultraviolet Photodissociation with Proton Transfer Reactions and Ion Parking for Analysis of Intact Proteins

We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case, a nitrogen-containing adduct of fluoranthene. Using PTR and ion parking, we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissociation (HCD) or ultraviolet photodissociation (UVPD). UVPD exhibited less charge state dependence, thus yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD. HCD resulted in especially prominent cleavages C-terminal to amino acids containing acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine.

Ultraviolet Photodissociation of Native Proteins Following Proton Transfer Reactions in the Gas Phase

The growing use of mass spectrometry in the field of structural biology has catalyzed the development of many new strategies to examine intact proteins in the gas phase. Native mass spectrometry methods have further accelerated the need for methods that can manipulate proteins and protein complexes while minimizing disruption of noncovalent interactions critical for stabilizing conformations. Proton-transfer reactions (PTR) in the gas phase offer the ability to effectively modulate the charge states of proteins, allowing decongestion of mass spectra through separation of overlapping species. PTR was combined with ultraviolet photodissociation (UVPD) to probe the degree of structural changes that occur upon charge reduction reactions in the gas phase. For protein complexes myoglobin·heme (17.6 kDa) and dihydrofolate reductase·methotrexate (19.4 kDa), minor changes were found in the fragmentation patterns aside from some enhancement of fragmentation near the N- and C-terminal regions consistent with slight fraying. After finding little perturbation was caused by charge reduction using PTR, homodimeric superoxide dismutase/CuZn (31.4 kDa) was subjected to PTR in order to separate overlapping monomer and dimer species of the protein that were observed at identical m/z values.

Determination of Collision Cross-Sections of Protein Ions in an Orbitrap Mass Analyzer

We demonstrate a method for determining the collision cross-sections (CCSs) of protein ions based on the decay rate of the time-domain transient signal from an Orbitrap mass analyzer. Multiply charged ions of ubiquitin, cytochrome c, and myoglobin were generated by electrospray ionization of both denaturing solutions and ones with high salt content to preserve native-like structures. A linear relationship between the pressure in the Orbitrap analyzer and the transient decay rate was established and used to demonstrate that the signal decay is primarily due to ion-neutral collisions for protein ions across the entire working pressure range of the instrument. The CCSs measured in this study were compared with previously published CCS values measured by ion mobility mass spectrometry (IMS), and results from the two methods were found to differ by less than 7% for all charge states known to adopt single gas-phase conformations.

Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

AbstractWe evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model. Graphical Abstractᅟ