RESUMEN
Electrophoretic and chromatographic sample preparations were compared and together detected the presence of some 600 types of protein products in human serum. Proteins from crude serum preseparated by ionic electrophoresis, chromatography, or a combination of both were analyzed. Proteins were digested with trypsin or chymotrypsin. Naturally occurring peptides were also collected by reversed-phase chromatography. The resulting peptides were identified by tandem mass spectrometry. The peptides were either desorbed by a laser from a metal chip into a quadrupole-time-of-flight mass spectrometer or ionized as an electro-spray from reversed-phase chromatography via a metal needle under voltage into an ion-trap mass spectrometer. All of the commonly known proteins associated with serum were detected, and the two mass spectrometers agreed on the identity of abundant serum proteins. Preseparation of serum proteins prior to digestion markedly enhanced the capacity to detect un-common proteins from blood. Electrophoretic- and chromatography-based experiments were found to be complementary. Many novel cellular proteins not previously associated with serum were recorded.
Asunto(s)
Proteínas Sanguíneas/química , Cromatografía/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , HumanosRESUMEN
The MALDI-TOF spectra of peptides from the sera of normal and myocardial infarction patients produced patterns that provided an accurate diagnostic of MI. In myocardial infarction, the spectral pattern originated from the cleavage of complement C3 alpha chain to release the C3f peptide and cleavage of fibrinogen to release peptide A. The fibrinogen peptide A and complement C3f peptide were in turn progressively truncated by aminopeptidases to produce two families of fragments that formed the characteristic spectral pattern of MI. Time course and inhibitor studies demonstrated that the peptide patterns in the serum reflect the balance of disease-specific-protease and aminopeptidase activity ex vivo.
Asunto(s)
Proteínas Sanguíneas/análisis , Infarto del Miocardio/sangre , Mapeo Peptídico/métodos , Secuencia de Aminoácidos , Análisis de Varianza , Western Blotting , Complemento C3/metabolismo , Complemento C3b/metabolismo , Biología Computacional/métodos , Interpretación Estadística de Datos , Bases de Datos Genéticas , Procesamiento Automatizado de Datos/métodos , Fibrinógeno/metabolismo , Humanos , Datos de Secuencia Molecular , Análisis Multivariante , Infarto del Miocardio/diagnóstico , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Fluoruro de Fenilmetilsulfonilo/farmacología , Inhibidores de Proteasas/farmacología , Proteómica/métodos , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de TiempoRESUMEN
The Zn(2+)- and Ca(2+)-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn(2+) and Ca(2+). Zn(2+) binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca(2+)-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn(2+)/Ca(2+)-dependent relocation of S100B.
