Mass Spectrometric Determination of Protein Ubiquitination.

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. This "tag" can be used to distinguish a ubiquitinated peptide from the unmodified version, and can be incorporated into automated database searching. Several tags are discussed, the GGK and LRGGK tags, resulting from complete and incomplete tryptic digestion of the protein, and the STLHLVLRLRGG tag from a gluC-digested protein.A ubiquitinated peptide has two N-termini-one from the original peptide and the other from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain, and any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, have not previously been reported. These ions can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

A novel proteolytic processing of prolysyl oxidase.

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.

Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the kappa-opioid receptor.

Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.

Iminohydantoin lesion induced in DNA by peracids and other epoxidizing oxidants.

The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-chloroperbenzoic acid (m-CPBA) and dimethyldioxirane (DMDO) as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT).(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification, suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism has been probed by oxidation of the monomeric species dGuo, dGMP, and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP, and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with [(18)O(2)]-m-CPBA and H(2)(18)O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring-opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker.

Mass spectrometric determination of protein ubiquitination.

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. The ubiquitinated peptide thus has two N-termini - one from the original peptide and one from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain. Any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, have not previously been reported. These ions can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face.

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169-75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Galpha binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Galpha substrate.

Redox regulation of RhoA.

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).

Improved protein identification through the use of unstained gels.

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.

Mass spectrometric determination of protein ubiquitination.

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. The ubiquitinated peptide thus has two N-termini- one from the original peptide and one from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain. Any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation.

Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O2), regulates Ras activity. We show that treatment of Ras with NO/O2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide (*NO2). We also provide evidence that the Ras Phe28 side-chain plays a key role in the formation of a NO/O2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O2-mediated Ras GNE, in which *NO2, formed by the reaction of NO with O2, generates a Ras Cys118 thiyl radical (Ras-S118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O2-mediated Ras GNE. According to this mechanism, reaction of NO with O2 produces *NO2. *NO2 then reacts with Ras to produce Ras-S118, which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(+)-DP) via the Phe28 side-chain. G(+)-DP is subsequently converted to a neutral radical, and can react with another *NO2 to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases.

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination.

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.

Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin).

The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.