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Background With the changes of diet and living style,the diabetes has become the major diseases affecting human health.Diabetic cataract is a common complication of diabetes. Objective The present study was to investigate the difference of lens proteomics between diabetic cataract and age related cataract using two dimensional electrophoresis (2-DE) and mass spectrometry in order to postpone happening of diabetic cataract and offer the effective approach to the prevention and therapy of diabetic cataract. Methods The lenses were obtained from 8 diabetic patients and 12 age-related cataract patients during the surgery to extract the protein by lysis and centrifugation.The lens proteins were separated using immobilized pH gradients 2-DE.Image analysis was carried out using PDQuest Advanced-8.0.1 software package.Significant difference of the crystallines was identified by matrixassisted laser adsorption/ionization time of-flight-mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprint combined with protein database. Results The maps of 2-DE showed that lens proteins of diabetic cataract and age related cataract were in the section of pH 5-9 with the relative molecular weight 14000-97000;while relative molecular weight of more abundant crystalline was localized at 20000-31000.About 3 differential protein spots were detected by image analysis software.Two crystallines,αB and βB1 crystallin,were identified using MALDI-TOF-MS.Conclusions Proteomic analysis of lens can be accomplished and the proteins can be well separated,moreover,differential proteins can be analyzed using 2-DE and mass spectrometry between diabetic cataract and age related cataract.These results indicate that αB and βB1 crystallin proteins accelerate the development of diabetic cataract.This technique offers a new avenue for clarity of lens proteins of diabetic cataract other than age related cataract.
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OBJECTIVE:To develop a high performance liquid chromatography-mass spectrography method (LC-MS/MS) for determination of huperzine A in human serum. METHODS:The clonidine was used as the internal standard,and the test serum samples after being liquid-liquid extracted by ethyl acetate,dried in organic phase and reconstituted in mobile phase were analyzed on a Phenomenex Luna CN column interfaced with LC-MS with Methanol-0.08% formic acid (46∶54 containing 0.2% ammonium acetate). Positive ion SRM detection was performed for ESI iron source. Target ions were at m/z 243. 2→210. 9 for Huperzine A with an impact energy of 30V and m/z 229. 9→212. 9 for the internal standard with an impact energy of 32V during 0.5 s (scan time). RESULTS:The assay was proved to be free from the interference from endogenous substance,and the linear concentration range of Huperzine A was 0.08～8 ng?mL-1(r=0.993 0～0.996 0). The intra-and inter-day precision over the entire concentration range were less than 15%. At low,middle and high concentrations,the mean extraction recoveries of Huperzine A were 69.9%,63.7% and 63.7%,respectively. CONCLUSION:The LC-MS/MS method is rapid,sensitive,simple,accurate and in which little amount of mediator is needed,thus meeting the requirement for the detection of biological specimen and applicable for the treatment of large batch of serum samples in clinical pharmacokinetic study.