Kevin M. Downard

Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the structure of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions using synchrotron light modify amino acid side-chains on millisecond timescales. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side-chains to the bulk solvent. The aromatic and sulphur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for a number of proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates correlate closely with a theoretical measure of the accessibility of residue side-chains to the solvent in the native protein structure. This approach can distinguish the relative accessibility of the tryptophan residue side-chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues, and phenylalanine at position 34 from phenylalanine residues at positions 3 and 38 based upon their rates of oxidation.

Electrospray-assisted modification of proteins: a radical probe of protein structure†

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a proteins higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.

Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins

The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a proteins three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin-melittin protein-peptide complexes are presented to illustrate the approach.

Preservation and detection of specific antibody—peptide complexes by matrix-assisted laser desorption ionization mass spectrometry

The direct detection of an antibody—peptide complex is reported by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Experimental conditions have been found in which specific, noncovalent interactions in solution are maintained throughout the sample preparation and ionization process. Mass measurements based on the ion signals for the intact antibody and 1:1 antibody—peptide complex reveal that specific noncovalent associations between a monoclonal antibody and a peptide, which comprises the determinant of the corresponding antigen, are maintained in the gas phase. These results support the wider application of MALDI-MS to studies of the structure and specificity of macromolecular complexes important to immune and other biological function.

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.

Subtyping of the influenza virus by high resolution mass spectrometry.

High resolution, high mass accuracy mass spectra of hemagglutinin and whole virus digests of influenza are shown to be able to be used to type and subtype the major circulating forms of the virus in humans. Conserved residues and peptide segments of the hemagglutinin antigen have been identified across type A and B strains, and for type B strains of the Yamagata 16/88 and Victoria 2/87 lineages. The theoretical masses for the protonated peptide ions for tryptic peptides of conserved sequence were subsequently shown to be unique in mass when compared to in silico generated peptides from all influenza viral protein sequences and those proteins known to contaminate virus preparations. The approach represents a more rapid and direct approach with which to type and subtype the virus that is of critical need to prepare strategies and treatments in the event of a local epidemic or global pandemic.

Contributions of mass spectrometry to structural immunology.

Mass spectrometry has made important contributions to the field of immunology in the past decade. A variety of mass spectrometric-based techniques have been applied to study the structures of macromolecules that play a vital role in the immune response. These include traditional molecular mass measurements to identify post-translational modifications and structural heterogeneity, mass mapping of proteolysis products, sequencing by tandem mass spectrometry and conformational analysis. Antigen-antibody and other immune complexes have been detected by mass spectrometry, providing an avenue to study macromolecular assemblies that are important to immune function. By virtue of the ability of mass spectrometry based techniques to analyze complex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopes important in both the humoral and cellular immune responses. This has been achieved through a combination of immunoaffinity and mass spectrometric techniques, and the coupling of high-performance chromatographs to mass spectrometers. These approaches are important for the identification of pathogens and show promise for the early diagnosis of disease associated with viral and bacterial infection and malignancy. These investigations will enable the mechanisms associated with normal and impaired immune function to be elucidated. Mass spectrometry has been utilized to characterize the structure of peptide mimics, multiple antigenic peptides and other constructs in the design of synthetic immunogens. Information derived from these studies will aid in the development of novel therapeutics and vaccines.

Signature peptides of influenza nucleoprotein for the typing and subtyping of the virus by high resolution mass spectrometry

The use of high resolution mass spectrometry to record the accurate mass of signature peptides within proteolytic digests of the nucleoprotein antigen, and whole influenza virus, is shown to be able to rapidly type and subtype the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of those for the nucleoprotein across all influenza types and subtypes. Peptides with unique theoretical masses from those generated in silico for all influenza antigen sequences, and from those proteins known to contaminate virus preparations in laboratory grown samples, were identified using a purpose built algorithm (FluGest). The frequency of occurrence of such conserved peptide signatures was assessed across all nucleoprotein sequences to subsequently type and subtype human strains of the virus. The application of the approach is illustrated for both type A H1N1 and H3N2, and type B strains of human influenza virus.

PROXIMO—a new docking algorithm to model protein complexes using data from radical probe mass spectrometry (RP-MS)

The design and implementation of a new algorithm, known as PROXIMO for protein oxidation interface modeller, is described to predict the structure of protein complexes using data generated in radical probe mass spectrometry (RP-MS) experiments. Photochemical radiolysis and discharge sources can be used to effect RP-MS in which hydroxyl radicals are formed directly from the bulk solvent on millisecond timescales and react with surface accessible residues in footprinting-like experiments. The algorithm utilizes a geometric surface fitting routine to predict likely structures for protein complexes. These structures are scored based on a correlation between the measured solvent accessibility of oxidizable residue side chains and oxidation shielding data obtained by RP-MS. The algorithm has been implemented to predict structures for the ribonuclease S-protein-peptide and calmodulin-melittin complexes using RP-MS data generated in this laboratory. The former is in close agreement with the high-resolution experimental structure available.

Amino acid sequence prerequisites for the formation of cn ions

Ammo acid sequence prerequisites are described for the formation of c, ions observed in high-energy collision-induced decomposition spectra of peptides. It is shown that the formation of cn ions is promoted by the nature of the amino acid C-terminal to the cleavage site. A propensity for cn cleavage preceding threonine, and to a lesser extent tryptophan, lysine, and serine, is demonstrated where fragmentation is directed N-terminally at these residues. In addition, the nature of the residue N-terminal to the cleavage site is shown to have little effect on cn ion formation. A mechanism for cn ion formation is proposed and its applicability to the results observed is discussed.