ABSTRACT
Objective To investigate human plasma glycoproteomie changes related to human immunodeficiency virus (HIV) infection,and to identify glycoproteins with potential anti-HIV activity or anti-HIV drug targets. Methods Plasma proteins with lower abundance were enriched through affinity purification to remove albumin and IgG in clinical samples (HIV-positive patient, n= 10, and healthy controls, n= 20). Proteins were separated by two-dimensional electrophoresis (2-DE) and stained by Pro-Q emerald glycoprotein stain kits. The 2-DE image was analyzed by ImageMaster software to find differential glycoproteins. Furthermore, the depleted HIV-positive and healthy control plasma proteins were digested by PNGase F. Glycoproteins were deglycoliszed, separated by 2-DE and analyzed by ImageMaster software. Differential glycoproteins were identified by liquid chromatography combined with high capacity ion trap mass spectrometry (HCT). Results The pretreatment of HIV-positive plasma prior to 2-DE could efficiently remove the high aboundant albumin and IgG in plasma and improve the detection of proteins with low-abundance. High revolution 2-DE gel images of glycoproteins from HIV positive and healthy control plasma samples were obtained. Glycoproteins were successfully deglycolized through PNGase F treatment. Thirteen differential glycoproteins were identified by liquid chromatography combined with mass spectrometry. These proteins included alphalantitrypsin precursor and serine/threonine-protein kinase N1. Conclusions Potential HIV infection related proteins,such as alphal-antitrypsin precursor are successfully identified. Our study may offer some help to understand the molecular mechanism of HIV infection and select new drug targets for preventing HIV infection.
ABSTRACT
ObjectiveTo find specific biomarkers related to HAART treatment in plasma samples of AIDS patients for clinical therapeautic efficacy evaluation and guidance for the prognosis of HIV treatment.MethodPlasma samples of AIDS patients were collected from Infectious Disease Department 1 of Shanghai Public Health Clinical Center in June of 2008 to February of 2009,including 11 successfully HAART treated cases (HIV load ＞ 50 copies/ml) and 11 unsuccessfully HAART treated cases (HIV load ＜50 copies/ml).Patients' age ranged from 22 to 63.Plasma samples were treated by Bio-rad AurumTM Serum Protein Mini Kit to remove high abundant proteins:albumin and immunoglobulin were removed.The treatedplasmaproteinswereseparatedbytwo-dimensionalelectrophoresisandanalyzedby electrophoretogram using Imagemaster software to find differentially-expressed proteins related to therapeutic efficacy.After digestion by trypsin,the differentially-expressed proteins were identified by online reversed-phasenano-flow liquid chromatography coupled with electrospray ionization ion trap mass spectrometry.ResultsLow abundant proteins were efficiently enriched after the AurumTM Serum Protein Mini Kit treatment.Six differentially-expressed proteins were detected while comparing successfully and unsuccessfully HAART treated group.These proteins were accurately identified by tandem Mass spectrometry (MS), including serum transferrin, serum β-fibrinogen, etc.ConclusionsOur proteomic research revealed that the differentially-expressed proteins such as transferrin,which is related to plasma virus loading in AIDS patients in the process of treatment,might be potential biomarkers evaluating HAART therapeutic efficacy.
ABSTRACT
A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of lopinavir and ritonavir in human plasma. Analytes were separated from plasma by a combination of alkalinized protein precipitation and liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Agilent ZORBAX Eclipse XDB-C18 column with the mobile phase consisted of methanol-0.1% formic acid in water (80:20). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 629.6 --> 155.2, m/z 721.4 --> 268.2, and m/z 515.2 --> 276.2 for lopinavir, ritonavir and telmisartan (internal standard), respectively. The method showed a good linearity in a concentration range of 62.5 - 10000 ng mL(-1) for lopinavir, and 12.5 - 2000 ng mL(-1) for ritonavir. The lower limits of quantification were 15 pg mL(-1) and 8 pg mL(-1) for lopinavir and ritonavir, respectively. The intra- and inter-day precision was less than 15% and the absolute recovery was above 75%. This method was selective and rapid, sensitive for investigating blood drug concentrations in clinics.