Attempt On Analyzing Proteins In CSF Of Parkinson Disease Patients With 2D-PAGE

Objective: Analyzing the global protein patterns in cerebrospinal fluid(CSF) of Parkinson Disease patients and controls (patients with headache) with two-dimensional polyacrylimide electrophoresis (2D-PAGE). Finding out the most appropriate experiment scenario to acquire better resolving effect. Screening out significant differentially displayed protein spots by comparing two groups of 2D-PAGE patterns and estimate their identities and functions.Methods: ① Collecting CSF samples by lumbar puncture from PD patients and controls. ② The concentration of proteins in CSF was measured by the method of Bradford and acetone was employed to precipitate the protein from the fluid. ③ Approximate 150μg proteins extracted from CSF was run immobilized pH gradient isoelectic focusing (IPG-IEF) as the first dimension, and then vertical flat sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension. ④ The protein spots in gels was visualized by silver staining protocol followed by gel scanning and image analysis. Searching for differentially displayed protein spots by comparing two groups of gel images and primarily analyzing them.Results: ① Satisfied 2D-PAGE patterns (pH3-10 and pH4-7) of protein in CSF of PD patients and controls was acquired in this experiment. Distributing of the 2D-PAGE pattern of CSF protein was similar to that in serum. Most of protein in CSF was neutral and their pI range from 4 to 8.The molecular mass of these protein mainly were between 20 and 100kD.A mass of protein congregated about 70kD and they were likely to be albumin from serum according to previous literature. ② Three gel images of PD group were compared to verify the reduplicativity of 2D-PAGE. 221, 232, 257 protein spots were detected in three gels,respectively. 30 protein spots matching each other from every gel respectively were analyzed and the results show that isoelectric point (pI) in three different experiments : 5.7562±0.3765,5.7587±0.3759,0.5962± 0.4553, F=0.19 (p>0.05, n=30); molecular mass(M) : 105.7754±6.8222kD,106.6466±7.515,107.4498±6.2455,F=0.341(p>0.05, n=30)；and volume of the same protein spots : 2893.1739±1430.3435,3116.5652±1431.4734,2723.7391±1461.4173,F=0.429(p>0.05 ,n=30). ③ Totally, 237±18 and 301±20 protein spots were detected in PD patients and controls CSF protein pH4-7 2D-PAGE map, respectively.8 spots increased and 9 spots decreased in quantity even 8 spots disappeared in PD patients CSF compared to controls. ④ It was postulated that spots 30,31,45 were ceruloplasmin(CP) and its precursor(CPP), spots 261,262,263,264,265 were β chains of haptoglobin(Hp) , spots 274,276,277,278,279,280,281,282,283 were fragments apolipoprotein J(Apo J) and its precursor according to their pI ,molecular mass and the reference map of human CSF in SWISS-2DPAGE database.⑤ After attempting several kinds of rehydration and sample application buffer comprised of different components, the 2D-PAGE patterns showed that DTT was able to resolve proteins better than TBP as a reducing agent,4%CHAPS,a zwitterionic detergent, was more effective than 2% ,and very SDS in very low concentration(0.25%) in the buffer can still bother IEF severely. Conclusion: High resolving ability and well-reduplicative protein patterns of human CSF were able to be acquired by the method of 2D-PAGE included of IPG-IEF and SDS-PAGE. Better protein patterns have been gained when the pH4-7 IPG dry strips were used comparing to pH3-10 strips. There were significant differences between CSF protein maps of PD patients and controls. Some protein spots increased or decreased in quantity even disappeared in CSF of PD patients compared to controls. The major subsequent work will be to identify these differentially displayed protein spots and learning their functions. In the same time, the resolving ability can be improved by choosing appropriate scenario of rehydration and sample application buffer as sample preparation was the key step of 2D-PAGE.