RESUMEN
N-glycan oligosaccharides of human serum alpha(1)-acid glycoprotein (AGP) samples isolated from 43 individuals (healthy individuals and patients with lymphoma and with ovarian tumor) were analyzed by MALDI-TOF mass spectrometry and a multivariate statistical method (linear discriminant analysis, LDA). 34 different glycan structures have been identified. From the glycosylation pattern determined by mass spectrometry fucosylation and branching indices have been calculated. These parameters show only small differences between the patient groups studied, but these differences are not sufficiently large to use as a potential biomarker. LDA analysis, on the other hand shows a very good separation between the three groups (with a classification of 88%). Cross-validation indicates that the method has predictive power: Identifying cancerous vs. healthy individuals shows 96% selectivity and 93% specificity; identification of lymphoma vs. the mixed group of healthy and ovarian tumor cases is also promising (72% selectivity and 84% specificity). The pilot study presented here demonstrates that mass spectrometry combined with linear discriminant analysis (LDA) may provide valuable data for identifying and studying the pathophysiology of malignant diseases.
Asunto(s)
Biomarcadores de Tumor , Orosomucoide/química , Polisacáridos/química , Análisis Discriminante , Femenino , Glicosilación , Humanos , Linfoma/sangre , Espectrometría de Masas , Orosomucoide/metabolismo , Neoplasias Ováricas/sangre , ProteómicaRESUMEN
A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).
Asunto(s)
Orosomucoide/química , Orosomucoide/metabolismo , Cromatografía Liquida , Glicosilación , Humanos , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
Chiral separation of D,L-Norgestrel was successfully performed by cyclodextrin-modified capillary electrochromatographic (CEC) method. Optimized CEC conditions for purity testing were achieved: Hypersil C-18 packed capillary and 10 mM 2-[N-morpholino]-ethanesulfonic acid buffer (MES) and 40 v/v % methanol as background electrolyte containing 10 mM gamma-cyclodextrin at pH = 5.5. The resolution was found to be strongly dependent on the concentration of chiral selector gamma-cyclodextrin, applied voltage and temperature of the capillary. Our results represent a simple, fast and reproducible method for the separation of D,L-Norgestrel and purity testing of D-Norgestrel. It can be concluded that cyclodextrin modified HPLC, HPCE and CEC offer inexpensive, attractive and reliable chiral separation methods.