RESUMEN
Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.
Asunto(s)
Antígenos/inmunología , Antígenos/metabolismo , Autoanticuerpos/inmunología , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/química , Autoanticuerpos/sangre , Dominio Catalítico , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Proteína Básica de Mielina/química , Especificidad por SustratoRESUMEN
The plasma peptide component (PPC) from ten melanoma (Mel), breast cancer (BC) and healthy individuals was examined by a combination of RP-HPLC, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and tandem mass spectrometry. A three peak pattern (2023, 2039, 2053.5 m/z) was primarily observed in melanoma. Two peaks (2236.1 and of 2356.3 m/z) were found only in BC samples. Fibrinogen alpha and inter-alpha-trypsin inhibitor heavy chain H4 fragments were absent in both tumor samples.
Asunto(s)
Neoplasias de la Mama/sangre , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Melanoma/sangre , Proteínas de Neoplasias/sangre , Péptidos/sangre , Proteómica/métodos , Neoplasias Cutáneas/sangre , Adulto , Anciano , alfa-Globulinas/análisis , Secuencia de Aminoácidos , Femenino , Fibrinógeno/análisis , Perfilación de la Expresión Génica , Humanos , Rayos Láser , MasculinoRESUMEN
The overall history and recent advancements in Surface-Enhanced Laser Desorption/Ionization (SELDI) affinity technology is reviewed. A detailed account of SELDI technology, utilizing Immobilized-Metal Affinity surfaces, pseudo-specific chromatographic surfaces, and biospecific interactive surfaces, is presented with particular emphasis placed upon examination of fundamental characteristics as well as specific applications for each. Finally, a detailed review of the specific use of such affinity surfaces in fundamental aspects of clinical, process, and research proteomics activity is presented.
Asunto(s)
Sondas Moleculares/química , Análisis por Matrices de Proteínas/métodos , Análisis por Matrices de Proteínas/tendencias , Proteínas/análisis , Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/tendencias , Técnicas de Sonda Molecular/tendencias , Sondas Moleculares/síntesis químicaRESUMEN
A novel analysis of explosives via the coupling of an airline passenger personnel portal with a high-flow (HF), high-resolution (HR) ion mobility spectrometry (IMS) was shown for the first time. The HF-HR-IMS utilized a novel ion aperture grid design with a (63)Ni ionization source while operating at ambient pressure in the positive ion mode at 200 degrees C. The HF-HR-IMS response characteristics of 2,4,6-trinitrotoluene (TNT), 4,6-dinitro-o-cresol (4,6DNOC), and cyclo-1,3,5-trimethylene-2,4,6-trinitramine (RDX) were investigated. Modifications made to the HF-HR-IMS exhaust and ionization source created an 800% increase in the total ion current (TIC), from 0.85 to 6.8 nA. This translated into a 65% ion response increase for TNT when compared with a traditional IMS. A mixture of TNT and (4,6DNOC) was used to successfully demonstrate the resolving power of the species with similar reduced mobility constants (K(o)), 1.54 and 1.59, respectively. The reactant ion (H(2)O)(n)H(+), peak was also used to measure the resolving power of the spectrometer while varying the internal diameter of three different aperture openings from 1.00 to 3.54cm. This provided a resolving power range of 50-60, double that typically achievable by commercial IMS instruments. Most important, these changes made in this new instrumental design can be implemented to all existing and future IMS's to greatly enhance the achievable IMS resolving power.