Analysis Serum Markers Of Hepatocellular Carcinoma HPX And HCâ…¡ By MRM Technology

HCC (hepatocellular carcinoma, HCC) is the highest incidence tumor in thedeveloping countries and the third killer of cancer-related death in the world. Atpresent, the incidence of HCC has been gradually rising in our country, themortality rate is also the highest in China. The occurrence of HCC is highlyrelated with hepatitis infection and liver fibrosis. As a result of the limitation ofexisting diagnosis and treatment technology, most of the HCC patients werealready in middle-late after diagnosis that lead to bad prognosis which also makethe treatment more difficult. If it can be early diagnosed and timely treatmentaccording to surgery, liver transplantation, interventional embolization and radiofrequency, it may has great therapeutic effective. As far as we know the clinicalserum markers of HCC is AFP, however the AFP has20%-30%of the falsepositive rate, there are still some HCC patients whose AFP is negative, so thebiomarkers of AFP negative HCC are the focus of currently research.In recent years, the high flux quantitative detection of stable isotopelabeling mass spectrometry provides a new technology platform for the researchof functional proteomics. In the previous study, a number of differentiallyexpressed proteins of HCC had been screened and identified by iTRAQtechnology. Due to the influence of gene’s instability and heterogeneity inhuman tumor, the proteins not only has the concomitant changes but also has thekey changes. So how to identify that key molecular changes from concomitant is one of the important challenges in the field of tumor marker research. To solvethis problem, the following strategies had been used:(1) the differentiallyexpressed proteins come form the comparison of HCC group with hepatitisgroup and the comparison of HCC group with liver fibrosis group;(2) Using thesoftware of STRING to get the key nodes in the construct of the proteininteraction networks. After analysis the key nodes of differentially expressedproteins,7candidate protein were got in addition to AFP, they were: Heparincofactor2(HCII), Angiotensinogen (AGT), Hemopexin (HPX), ComplementC4-B (C4B), Insulin-like growth factor-binding protein3(IGFBP3), Fibronectin(FN1) and Gelsolin (GSN).The target protein quantitative technology which based on massspectrometry is the bridge of the markers from finding to the clinical verification,and the multiple reaction monitoring (multiple reaction monitoring, MRM)technology has become the focus which with the advantages of one-timesimultaneously detect more than one candidate proteins, high specificity andhigh sensitivity data. The mass spectrometer can monitoring a large quantities ofsamples by MRM scan mode to see whether the pair ions were appeared when itwas analyzing. If the signals appeared which prove the existence of themolecules in the sample, the instrument will automatically switch to the MS/MSscan mode to detect the fragments of the pair ions information and verify thestructure of the molecule again. With the high efficiency and high sensitivity ofthe scan mode, it’s very suitable for the verification of biomarkers. what’smore,the characteristics also offers a condition to quantitative proteins at onetime.Seven target protein expression map have been got when using theiTRAQ technology at the early experiment. And MRM as another moresensitive mass spectrum detection technology was used this time to detect the proteins again especially focused on the protein Hemopexin(HPX) and Heparincofactors Ⅱ (HCII) to verify the consistency between MRM and iTRAQtechnology’s result, and the reference of AFP negative HCC patients’ newbiomarker will be find according to this research.Chapter one Using MRM technology to establish the massspectrometry relative quantitative method.OBJECTIVE Using the MRM (multiple reaction monitoring, MRM)technology to establish a mass spectrometry quantitative detection methodwhich is not rely on antibodyMETHODS The standard substance BG (Beta-Galactosidase, BG)quantitative peptides were chosen and the parent ion pair were optimized whichwas chosen from the peptides by the skyline software. After five repeated trials,the RSD%(Relative Standard Deviation) results have been obtained. In order to imitate serum complex proteins environment, BG and the yeast extract enzymeproducts were mixed to establish a standard curve under complex background.RESULTS The three unique peptides of BG for the protein quantitativeanalysis which identified many times in the experiment were: VDEDQPFPAVPK, IDPNA WVER, GDFQFNISR. After5times trials, the RSD%results ofthe integral peak area were less than6%at each time which proved the precisionof the method. The three peptides showed good linear correlation within thescope of0.625-50×10-6fmol/L, and the R2value of standard curve were0.99852-0.99955. It could still be detected stability mass peak even the BG was lowerthan0.625×10-6fmol/L, which shows the high sensitivity of the method.CONCLUSION The relative quantitative method was established to detect BG by using the MRM mass spectrometry. It will be the foundation for theserum markers research.Chapter two Analysis of potential serum markers HPX andHCII of HCC by relative quantitative MRM methodOBJECTIVE Using the established MRM technology to detect HCC patientsserum HPX and HCII protein expression level and verify the iTRAQ results.METHODS1.10cases of AFP negative HCC serum were matched with10cases of normalcontrol group serum. After the high abundance ratios protein of serumsamples were removed, the serum was enzymolysis, iTRAQ labeled, SCXfractioned and reverse C18column eluted,finally the5800-MALDI-TOF/TOFmass spectrometry was used to detect the samples.2. Serum of5AFP negative HCC patients which without the anti-tumortreatment were1:2matched with the serum of10healthy control patients bygender and age. The selection of HPX and HCII two quantitative proteinpeptides were optimized according to the CE by skyline software.Then thesamples of HPX and HCII two protein expression level were detected byMRM technology.RESRLTS1. The HPX expression level in serum of AFP negative HCC patients detectedby iTRAQ combined LC-MALDI-TOF/TOF mass spectrometry was116/113=3.5645, P>0.05; and HCII expression level decreased in AFPnegative HCC patients which was116/113=0.7178, P=0.0179.2. The protein peptides that can be only used in quantitative analysis identifiedof HPX by MRM technology were EWFWDLATGTMK, GGYTLVSGYPK, NFPSPVDAAFR; and HCII were QFPILLDFK,TLEAQLTPR,YEITTIHNLFR.3. BG standard was joined to calibrate the system error. After comparing thepeak areas between the AFP negative HCC patients and normal group, theresults showed that the HPX peak area ratio in normal group and AFPnegative HCC patients was431.58±116.74and347.62±84.89(P>0.05)respectively;and HCII was26.604±11.645and16.032±4.381(P <0.05)respectively, HCII expressed decreased in AFP negative HCC patients.4. The MRM result was consistent with iTRAQ results on relative quantitativedetection of protein HPX and HCII.CONCLUSION1.The multiple protein markers could be detected at one time by MRMtechnology.2.The expression of HCII in AFP negative HCC patients were decreasedconfirmed by MRM results, it may be the AFP negative serum marker ofHCC patients.3.MRM is a simple operation, high sensitivity, high accuracy, goodreproducibility method, it has a unique advantages for testing multiplecandidate proteins. It is a reliable qualitative and quantitative method forverifing serum markers of HCC.