RESUMEN
The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARGS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARGS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARGS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.
Asunto(s)
Agrecanos/análisis , Líquidos Corporales/química , Cartílago Articular/patología , Cromatografía Liquida/métodos , Endopeptidasas/metabolismo , Espectrometría de Masas en Tándem/métodos , Agrecanos/química , Agrecanos/orina , Secuencia de Aminoácidos , Animales , Biomarcadores/análisis , Cartílago Articular/enzimología , Bovinos , Cromatografía de Afinidad , Perros , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/orina , Ratas , Estándares de Referencia , Reproducibilidad de los Resultados , Líquido Sinovial/metabolismo , Investigación Biomédica TraslacionalRESUMEN
OBJECTIVE: Progression of joint damage in osteoarthritis (OA) is likely to result from an imbalance between cartilage degradation and synthesis processes. Markers reflecting these two components appear to be promising in predicting the rate of OA progression. Both N- and C-terminal propeptides of type II collagen reflect the rates of collagen type II synthesis. The ability to quantify the procollagen peptides in biological fluids would enable a better understanding of OA disease pathology and provide means for assessing the proof of mechanism of anabolic disease modifying OA drugs (DMOADs). METHODS: A polyclonal antibody that recognizes the sequence GPKGQKGEPGDIKDI in the propeptide region of rat, dog, and human type II collagen was raised in chicken and peptide-affinity purified. The immunoaffinity liquid chromatography mass spectrometry (LC-MS/MS) was used to extensively characterize N-terminal procollagen type II (NPII) peptides found in biological fluids. The novel competition enzyme-linked immunosorbent assay (ELISA) assay was developed to quantitatively measure the NPII peptides. RESULTS: Several peptides ranging from 17 to 41 amino acids with various modifications including hydroxylations on proline and lysine residues, oxidation of lysines to allysines, and attachments of glucose and galactose moieties to hydroxylysines were identified in a simple system such as ex vivo cultures of human articular cartilage (HAC) explants as well as in more complex biological fluids such as human urine and plasma. A competitive ELISA assay has been developed and applied to urine, plasma, and synovial fluid matrices in human, rat and dog samples. CONCLUSION: A novel NPII assay has been developed and applied to OA and normal human subjects to understand the changes in collagen type II synthesis related to the pathology of OA.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno Tipo II/biosíntesis , Osteoartritis/metabolismo , Procolágeno/biosíntesis , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Cromatografía Liquida , Progresión de la Enfermedad , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Fragmentos de Péptidos , RatasRESUMEN
Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno Tipo II/química , Epítopos/análisis , Metaloproteinasas de la Matriz/metabolismo , Anciano , Secuencia de Aminoácidos , Biomarcadores/análisis , Células Cultivadas , Cromatografía Liquida/métodos , Colágeno Tipo II/metabolismo , Colagenasas/metabolismo , Femenino , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fragmentos de Péptidos/químicaRESUMEN
OBJECTIVE: To examine whether urine concentrations of type II collagen neoepitope (uTIINE) distinguish subjects with progressive radiographic and/or symptomatic knee osteoarthritis (OA) from those with stable disease. METHODS: Subjects were 120 obese middle-aged women with unilateral knee OA who participated in a 30-month randomized-controlled trial of structure modification with doxycycline, in which a standardized semiflexed anteroposterior view of the knee was obtained at baseline, 16 months and 30 months. Subjects were selected from a larger sample to permit a priori comparisons between 60 OA progressors and 60 nonprogressors, as defined by joint space narrowing (JSN) in the medial tibiofemoral compartment. Each group contained 30 subjects who exhibited clinically significant increases in knee pain over 30 months and 30 who did not. Urine samples were obtained every 6 months for determination of the creatinine (Cr)-adjusted uTIINE concentration. RESULTS: Baseline uTIINE levels were unrelated to JSN in the placebo group. However, among subjects in the active treatment group, a 1-standard deviation increment in baseline uTIINE (68 ng/mM Cr) was associated with a marginally significant, two-fold increase in the odds of progression of JSN (odds ratio 2.04, 95% confidence interval 0.98-4.28). The within-subject mean of uTIINE values at baseline, 6 months and 12 months was associated with concurrent JSN measured at 16 months (0.10mm of JSN per 69 ng/mM Cr, P=0.008). Similar results were seen in the interval between months 16 and 30 and in analyses using the maximum of intercurrent uTIINE levels. CONCLUSION: Baseline uTIINE was not a consistent predictor of JSN in subjects with knee OA. However, serial measurements of uTIINE reflect concurrent JSN.
Asunto(s)
Colágeno Tipo II/orina , Epítopos/orina , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/orina , Antibacterianos/uso terapéutico , Biomarcadores/orina , Índice de Masa Corporal , Colágeno Tipo II/inmunología , Creatina/orina , Doxiciclina/uso terapéutico , Epítopos/inmunología , Femenino , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/inmunología , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/inmunología , Obesidad/orina , Osteoartritis de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/tratamiento farmacológico , Valor Predictivo de las Pruebas , Radiografía , Reproducibilidad de los ResultadosRESUMEN
We studied by tandem mass spectrometry two isomers of nitro meso-tetraphenylporphyrin, one with a nitro group in the para position of a phenyl ring and the other with the same group in a beta-pyrrolic position, and their copper complexes. Collisional activation of the molecular ions of both free-base porphyrins and of their copper complexes produces an array of product ions that permit ready differentiation of the two positional isomers. The diagnostic ions, when the nitro group is in a beta-pyrrolic position, may be produced through intramolecular and double cyclization processes, triggered by the interaction of the nitro substituent with the neighboring meso-phenyl ring. These diagnostic ions do not form when the nitro group is in the para position. The gas-phase processes have precedents in solution chemistry.
Asunto(s)
Porfirinas/química , Cobre/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
We extended the kinetic method to determine the intrinsic affinities of nonvolatile organic molecules with divalent metal ions and then applied the amended method to determine the calcium affinities of peptide analogs of the calcium-binding site III of rabbit skeletal troponin C. Metal-bis(peptide) complexes of the composition ([H2Pi + H2Pii] - H + Ca)+, where H2P is a neutral peptide, were introduced into the gas phase by fast atom bombardment. The extended kinetic method recognizes that the dissociation characteristics of a singly charged, bis(peptide) complexes of divalent metal ions are determined by not only the metal-ion affinity but also the proton affinities of the neutral and deprotonated peptides. The modified method requires one to measure the relative abundances of [H2P - H + Ca]+, [H2P + H]+, and [H2P - H]- ions that form upon collisional activation of mixed peptide/metal complexes, proton-bound peptide dimers, and deprotonated peptide dimers, respectively. We found, by using the modified method, that the set of peptides has a different affinity order than that in solution. Peptides with one aspartic acid have a higher intrinsic Ca2+ affinity than those with two aspartates. The location of the aspartic acid (Asp) residues at various positions also affects the Ca2+ affinity. Those peptides with one Asp in the middle of the chain have higher Ca2+ affinities than those with Asp on the end because the former peptides offer greater polarizability to stabilize the charge. Peptides with two Asp's located in close proximity have higher intrinsic calcium affinities than those with aspartates positioned further apart.
Asunto(s)
Calcio/química , Músculo Esquelético/química , Oligopéptidos/química , Troponina C/química , Animales , Cinética , Espectrometría de Masas , Músculo Esquelético/metabolismo , Unión Proteica , Conejos , Troponina C/metabolismoRESUMEN
The precursor ions of tetraphenylporphyrins that are substituted with fatty acids can be introduced into the gas phase by matrix-assisted laser desorption ionization (MALDI) and undergo post-source and collisionally activated decompositions (CAD) in a time-of-flight mass spectrometer. The goal of the research is to obtain a better understanding of post-source decompositions (PSD); specifically, we asked the question of whether ions undergoing PSD have sufficient energy to give charge-remote fragmentations along an alkyl chain. We chose the porphyrin macrocycle because we expected it to act as an inert "support," allowing the molecule to be desorbed by MALDI and to be amenable to charge-remote fragmentation. MALDI-PSD and MALDI-CAD spectra are similar to high-energy CAD spectra and considerably more informative than low-energy CAD spectra, showing that charge-remote fragmentations of the fatty acid moieties do occur upon MALDI-PSD and MALDI-CAD.
Asunto(s)
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ácidos Grasos/química , Indicadores y Reactivos , Porfirinas/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Gas-phase complexes of cysteine-containing peptides and Fe2+ were produced by fast atom bombardment and studied by tandem mass spectrometry. Specific and strong interactions of the iron and sulfur from the thiol group of the cysteine side chain are preserved in the gas phase and are the basis for highly specific fragmentation to give abundant [a(n) - 2H + Fe]+ ions, where n is position of the cysteine residue from the N-terminus of peptide. Metal/peptide complexes containing more than one Cys residue were also investigated; they display similar chemistry upon collisionally activated decompositions, indicating that the Fe2+ ion primarily binds at cysteine sites.
Asunto(s)
Cisteína/química , Hierro/química , Péptidos/química , Sitios de Unión , Indicadores y Reactivos , Oligopéptidos/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.
Asunto(s)
Proteínas de Unión al Calcio/química , Péptidos/química , Sitios de Unión , Gases , Indicadores y Reactivos , Espectrometría de Masas , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
High-energy (HE) and low-energy (LE) collisionally activated decompositions of octaethylporphyrin (OEP) and its metal complexes (ZnOEP and CuOEP) depend on whether the precursor is produced by electrospray ionization as protonated molecules or by fast atom bombardment as radical cations or protonated molecules. LE activation leads to such simple product-ion spectra that a complete picture of fragmentation emerges only after nine stages of tandem mass spectrometry (MS). HE activation, on the other hand, gives product-ion spectra that afford an integrated view of all the decomposition channels in a single MS/MS experiment. These results are the basis of a recommendation that OEP is an appropriate model compound for investigating energy effects in the collisional activation of organic and bioorganic molecule ions.
Asunto(s)
Metales/análisis , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Gas-phase interactions of peptides that contain cysteine with iron(II) atoms were examined by using fast-atom bombardment and tandem mass spectrometry. Specific and strong interactions of iron and sulfur from the thiol group of the cysteine side chain occur in the gas phase and are the basis for highly specific fragmentation to give abundant [a n -(+) ions. For peptides that contain two cysteines, an internal ion, which results from the interaction of Fe and both thiol groups, is formed upon collisional activation. The mechanism for the formation of [a n -2H+Fe](+) fragment ions requires the metal to be coordinated at sulfur in close proximity to the site of reaction. Iron-bis(pentapeptide) complexes, which form under the same conditions, decompose predominantly to lose a pentapeptide molecule and, to a lesser extent, to give [a a -2H+Fe](+) ions.