Ryan R. Julian
University of California, Riverside
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Ryan R. Julian.
Journal of Proteome Research | 2009
Qingyu Sun; Hosea M. Nelson; Tony Ly; Brian M. Stoltz; Ryan R. Julian
A crown ether based, photolabile radical precursor which forms noncovalent complexes with peptides has been prepared. The peptide/precursor complexes can be electrosprayed, isolated in an ion trap, and then subjected to laser photolysis and collision induced dissociation to generate hydrogen deficient peptide radicals. It is demonstrated that these peptide radicals behave very differently from the hydrogen rich peptide radicals generated by electron capture methods. In fact, it is shown that side chain chemistry dictates both the occurrence and relative abundance of backbone fragments that are observed. Fragmentation at aromatic residues occurs preferentially over most other amino acids. The origin of this selectivity relates to the mechanism by which backbone dissociation is initiated. The first step is abstraction of a beta-hydrogen from the side chain, followed by beta-elimination to yield primarily a-type fragment ions. Calculations reveal that those side chains which can easily lose a beta-hydrogen correlate well with experimentally favored sites for backbone fragmentation. In addition, radical mediated side chain losses from the parent peptide are frequently observed. Eleven amino acids exhibit unique mass losses from side chains which positively identify that particular amino acid as part of the parent peptide. Therefore, side chain losses allow one to unambiguously narrow the possible sequences for a parent peptide, which when combined with predictable backbone fragmentation should lead to greatly increased confidence in peptide identification.
International Journal of Mass Spectrometry | 2001
Ryan R. Julian; J. L. Beauchamp
The binding of 18-crown-6-ether (18C6) to biologically relevant groups is studied by way of electrospray ionization (ESI) quadrupole ion-trap mass spectrometry. Isolated lysine is a special case in which the 18C6 attaches to the N-terminus, deriving stability from the resulting salt-bridge structure. In a peptide, 18C6 forms a stable supramolecular adduct with lysine by way of specific hydrogen bonding with the primary protonated amine on the side chain. 18C6 will bind to adjacent lysines, forming multiply charged complexes. For example, the most intense peak in the spectrum of a mixture of 18C6 and tetralysine is the quadruply charged peptide with four crown ethers attached. Competitive binding of 18C6 by other basic sites such as the n-terminus, histidine, and arginine limits the utility of this technique for the molecular recognition of lysine. Adducts of 18C6 with proteins such as cytochrome-c (CytC) and bovine pancreatic trypsin inhibitor (BPTI) reveal structural information relevant to the accessibility of potential binding sites. Adduct formation with 18C6 appears to increase the surface activity of peptides in a charged droplet and inhibits deprotonation during ion desolvation in the ESI process. These effects increase both the primary charge state and ion abundance of the analyte. Sequestering and stabilization of charge provided by 18C6 allows for the detection of species that are normally difficult to observe using ESI, such as glycine. The relationship between the observed behaviors of 18C6 adducts and the proposed ion evaporation mechanism of ESI is discussed.
Angewandte Chemie | 2009
Tony Ly; Ryan R. Julian
Unraveling of all of the information contained in proteomes poses a tremendous chemical challenge, which is balanced by the promise of potentially transformational knowledge. Mass spectrometry offers an unprecedented arsenal of tools for diverse proteomic investigations. Recently, it was demonstrated that ultraviolet light can be utilized to initiate unique and potentially useful fragmentations in peptides and proteins. Either nonspecific dissociation or highly specific dissociation at engineered chromophoric sites is possible following photon absorption. The level of specificity and control over fragmentation in these experiments is greater than with other fragmentation methods. Novel techniques made possible by this technology are poised to make substantial contributions to the field of proteomics.
Journal of the American Chemical Society | 2010
Tony Ly; Ryan R. Julian
A new method for identifying residue specific through space contacts as a function of protein secondary and tertiary structure in the gas phase is presented. Photodissociation of a non-native carbon-iodine bond incorporated into Tyr59 of ubiquitin yields a radical site specifically at that residue. The subsequent radical migration is shown to be highly dependent on the structure of the protein. Radical-directed dissociation (RDD) of low charge states, which adopt compact structures, generates backbone fragmentation that is prominently distributed throughout the protein sequence, including residues that are distant in sequence from Tyr59. Higher charge states of ubiquitin, which adopt elongated, unfolded structures, yield RDD that is primarily nearby in sequence to Tyr59. Regardless of which structure is probed, information at the residue-level is obtained by examining specific radical-donor and radical-acceptor pairs. The relative importance of a particular interaction pair for a specific conformation can be revealed by tracking the charge state dependence of the dissociation. Structurally important contact pairs exhibit strong and concerted changes in relative intensities as a function of charge state and can also be used to reveal structural features which persist among different protein structures. Moreover, incorporation of distance constraint information into molecular mechanics conformational searches can be used to drive the search toward relevant conformational space. Implementation of this approach has revealed highly stable, previously undiscovered structures for the +4 and +6 charge states of ubiquitin, which bear little resemblance to the crystal structure.
Analytical Chemistry | 2011
Arun Agarwal; Jolene K. Diedrich; Ryan R. Julian
Disulfide bonds stabilize the tertiary and quaternary structure of proteins. Identifying the correct disulfide bond pairs can be extremely useful to understand the nature of a protein. However identifying correct disulfide linkages remains a challenge for many proteins. We report the use of ultraviolet photodissociation (UVPD) at 266 nm to selectively cleave disulfide bonds in the gas phase, while leaving all other bonds intact. This methodology can be used to identify disulfide bonded pairs in complex systems with multiple disulfide bond partners. We have explored UVPD chemistry on pairs of model peptides with one disulfide bond to evaluate the importance of various sequence and structural effects. In addition, online experiments were performed on whole protein digests. Bond selective UVPD was able to correctly identify and characterize all known disulfide bonded pairs. The method also proved sufficiently sensitive to identify and characterize several non-native disulfide-bound peptide pairs which were present in trace amounts. Photodissociation at 266 nm can be a valuable tool for disulfide bond identification and pair assignment in high-throughput proteomics studies.
Journal of the American Chemical Society | 2008
Jolene K. Diedrich; Ryan R. Julian
Site-specific fragmentation of peptides at phosphorylated serine or threonine residues is demonstrated. This radical directed cleavage is accomplished by a two-step procedure. First the phosphate is replaced with naphthalenethiol using well established Michael Addition chemistry. Second, the modified peptide is electrosprayed and subjected to irradiation at 266 nm. Absorption at naphthalene causes homolytic cleavage of the connecting carbon-sulfur bond yielding a radical in the beta-position. Subsequent rearrangement cleaves the peptide backbone yielding a d-type fragment. This chemistry is generally applicable as demonstrated by experiments with several different peptides. Assignment of phosphorylation sites is greatly facilitated by this approach, particularly for peptides containing multiple serine or threonine residues.
Journal of the American Society for Mass Spectrometry | 2009
Tony Ly; Ryan R. Julian
Photodissociation of iodo-tyrosine modified peptides yields localized radicals on the tyrosine side chain, which can be further dissociated by collisional activation. We have performed extensive experiments on model peptides, RGYALG, RGYG, and their derivatives, to elucidate the mechanisms underlying backbone fragmentation at tyrosine. Neither acetylation nor deuteration of the tyrosyl phenolic hydrogen significantly affects backbone fragmentation. However, deuterium migration from the tyrosyl β carbon is concomitant with cleavage at tyrosine. Substitution of tyrosine with 4-hydroxyphenylglycine, which does not have β hydrogens, results in almost complete elimination of backbone fragmentation at tyrosine. These results suggest that a radical situated on the β carbon is required for a-type fragmentation in hydrogen-deficient radical peptides. Replacement of the αH of the residue adjacent to tyrosine with methyl groups results in significant diminution of backbone fragmentation. The initial radical abstracts an αH from the adjacent amino acid, which is poised to “rebound” and abstract the βH of tyrosine through a six-membered transition-state. Subsequent β-scission leads to the observed a-type backbone fragment. These results from deuterated peptides clearly reveal that radical migration in peptides can occur and that multiple migrations are not infrequent. Counterintuitively, close examination of all experimental results reveals that the probability for fragmentation at a particular residue is well correlated with thermodynamic radical stability. A-type fragmentation therefore appears to be most likely when favorable thermodynamics are combined with the relevant kinetic control. These results are consistent with ab initio calculations, which demonstrate that barriers to migration are significantly smaller in magnitude than probable dissociation thresholds.
Analytical Chemistry | 2010
Jolene K. Diedrich; Ryan R. Julian
Described herein are several unique analytical applications utilizing mass spectrometry and the selective modification of the free thiol form of cysteine in both peptides and proteins by various quinones. This simple modification can be used to quantify the number of free or disulfide bound cysteines in a protein. In addition, quinone modification can also be used to easily probe the solvent accessibility of cysteine residues, which provides information about protein structure or folding state. Furthermore, the chromophoric properties of the quinone moiety can be leveraged for site specific photodissociation of the backbone. The photodissociation reveals both the presence and location of modified cysteine residues. For example, cleavage of the protein backbone of alpha-hemoglobin is observed selectively at a single cysteine out of 140 residues in the whole protein. This selective backbone fragmentation is accompanied by a parent ion mass loss, which is unique to the modifying quinone. When combined, this information can be used to determine both the presence and site of modification generated by naturally occurring molecules, such as dopamine, which can harness quinone chemistry to modify proteins.
Chemical Communications | 2009
Benjamin N. Moore; Stephen J. Blanksby; Ryan R. Julian
Ion-molecule reactions between molecular oxygen and peptide radicals in the gas phase demonstrate that radical migration occurs easily within large biomolecules without addition of collisional activation energy.
Journal of the American Society for Mass Spectrometry | 2008
Tony Ly; Ryan R. Julian
The metal binding properties of proteins are biologically significant, particularly in relationship to the molecular origins of disease and the discovery of therapeutic pharmaceutical treatments. Herein, we demonstrate that selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) is a sensitive technique to investigate the structural effects of protein-metal interactions. We utilize specific, noncovalent interactions between 18-crown-6 ether (18C6) and lysine to probe protein structure in the presence and absence of metal ions. Application of SNAPP-MS to the calmodulin-Ca2+ system demonstrates that changes in protein structure are reflected in a substantial change in the number and intensity of 18C6s, which bind to the protein as observed by MS. In this manner, SNAPP is demonstrated to be a sensitive technique for monitoring ligand-induced conformational rearrangements in proteins. In addition, SNAPP is well-suited to examine the properties of natively unfolded proteins, where structural changes are more difficult to detect by other methods. For example, α-synuclein is a protein associated in the pathology of Parkinson’s disease, which is known to aggregate more rapidly in the presence of Al3+ and Cu2+. The 18C6 SNAPP distributions for α-synuclein change dramatically in the presence of 3 µM Al3+, revealing that Al3+ binding causes a significant change in the conformational dynamics of the monomeric form of this disordered protein. In contrast, binding of Cu2+ does not induce a significant shift in 18C6 binding, suggesting that noteworthy structural reorganizations at the monomeric level are minimal. These results are consistent with the idea that the metal-induced aggregation caused by Al3+ and Cu2+ proceed by independent pathways.