Zhaohui Sunny Zhou

Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry

A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.

A naturally occurring brominated furanone covalently modifies and inactivates LuxS

Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).

Enzyme-catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: a tool for the functional analysis of methyltransferases.

S-adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analogue functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analogue displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analogue expands the toolbox available to interrogate the biochemical functions of methyltransferases.

Protein Isoaspartate Methyltransferase-Mediated 18O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis

Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with (18)O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in (18)O-water to regenerate isoAsp. The specific replacement of (16)O with (18)O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the β-delta sleep-inducing peptide (DSIP) are demonstrated.

Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes

Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides, or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this posttranslational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin toward Hcy TL were observed.

Discovery of undefined protein cross-linking chemistry: a comprehensive methodology utilizing 18O-labeling and mass spectrometry.

Characterization of protein cross-linking, particularly without prior knowledge of the chemical nature and site of cross-linking, poses a significant challenge, because of their intrinsic structural complexity and the lack of a comprehensive analytical approach. Toward this end, we have developed a generally applicable workflow-XChem-Finder-that involves four stages: (1) detection of cross-linked peptides via (18)O-labeling at C-termini; (2) determination of the putative partial sequences of each cross-linked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search; (3) extension to full sequences based on protease specificity, the unique combination of mass, and other constraints; and (4) deduction of cross-linking chemistry and site. The mass difference between the sum of two putative full-length peptides and the cross-linked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible cross-linking chemistry and site were inferred, and ultimately confirmed, by matching with all data. Applying our approach to a stressed IgG2 antibody, 10 cross-linked peptides were discovered and found to be connected via thioethers originating from disulfides at locations that had not been previously recognized. Furthermore, once the cross-link chemistry was revealed, a targeted cross-link search yielded 4 additional cross-linked peptides that all contain the C-terminus of the light chain.

Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry.

In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via beta-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 degrees C, pH 7.0-9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine-containing peptides in the presence of a phosphine (tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H(2)O or D(2)O, suggested a radical-based desulfurization mechanism unrelated to beta-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis.

Discovery and Characterization of a Photo-Oxidative Histidine-Histidine Cross-Link in IgG1 Antibody Utilizing 18O-Labeling and Mass Spectrometry

A novel photo-oxidative cross-linking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder, 18O labeling and mass spectrometry (Anal. Chem.2013, 85, 5900−590823634697). Its structure was elucidated by peptide mapping with multiple proteases with various specificities (e.g., trypsin, Asp-N, and GluC combined with trypsin or Asp-N) and mass spectrometry with complementary fragmentation modes (e.g., collision-induced dissociation (CID) and electron-transfer dissociation (ETD)). Our data indicated that cross-linking occurred across two identical conserved histidine residues on two separate heavy chains in the hinge region, which is highly flexible and solvent accessible. On the basis of model studies with short peptides, it has been proposed that singlet oxygen reacts with the histidyl imidazole ring to form an endoperoxide and then converted to the 2-oxo-histidine (2-oxo-His) and His+32 intermediates, the latter is subject to a nucleophilic attack by the unmodified histidine; and finally, elimination of a water molecule leads to the final adduct with a net mass increase of 14 Da. Our findings are consistent with this mechanism. Successful discovery of cross-linked His-His again demonstrates the broad applicability and utility of our XChem-Finder approach in the discovery and elucidation of protein cross-linking, particularly without a priori knowledge of the chemical nature and site of cross-linking.

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

Zoosporic plant pathogens produce bacterial autoinducer-2 that affects Vibrio harveyi quorum sensing

The frequent coisolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore-free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate the production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from Phytophthora nicotianae, Phytophthora sojae, and Pythium aphanidermatum triggered luminescence of the Vibrio harve7yi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. The production of AI-2 by zoospores was confirmed by chemical assays. These results provide a new insight into the physiology and ecology of oomycetes.