Plasma Peptide Technology For The Separation And Screening Of Biomarkers For Lung Squamous Cell Carcinoma

Objective:1. Seek the test method and solution proportioning scheme that couldmaximize the extraction and separation of plasma polypeptide through the plasma samplingand polypeptide separating.2. Demonstrate the polypeptide sequence of healthy group andthat of the disease groups, and identify it; compare and analyze the polypeptide differencebetween the normal subjects and the patient with lung cancer to preliminarily screen theserum marker of lung squamous cell carcinoma.Method: Collect48cases of serum from healthy person,10cases of Phase I NSCLCspecimen,18cases of Phase II NSCLC patient and25cases of Phase III NSCLC patientusing P100test tube (BD Company, America) with stabilizing plasma. Separatepolypeptide using organic solvent precipitation and HPLC and demonstrate polypeptidegroup using MALDI-TOF-MS. Perform polypeptide sequencing on each group of samplethrough ESI-Q-TOF-MS, search protein or polypeptide sequence in the NCBI protein (gene)database using MASCO search engine and set those with the scoring of more than20asqualified ID sequence. So that obtain the plasma polypeptide group of the patient with lungcancer and that of healthy person and then compare the difference between the polypeptidegroup of patient with lung cancer and that of the healthy person.Results: We have performed detailed comparison and analysis on several differentcollection methods of plasma polypeptide group. As the lung cancer sample is difficult tobe collected, all plasmas used in the method study are collected from normal subjects.According to literature, we performed the heat-deactivated with high pressurized gelatinfiltering collection; the acetonitrile denaturized precipitation with high pressurized gelatinfiltering collection; and the acetonitrile/acetone denaturized precipitation with high pressurized gelatin filtering collection. After comparing these three methods by experiment,we found that organic solvent denaturized precipitation is superior to heat-inactivatedmethod, so we chose organic solvent precipitation as the prioritized processing method. Fororganic solvent precipitation, by comparing with the precipitation of different volumemultiples of acetonitrile, we found that acetonitrile/acetone/water precipitation in4timesof volume is superior to the acetonitrile precipitation in2.5times of volume, so we finallydetermined to use the acetonitrile/acetone/water precipitation as the extraction method forpolypeptide group. Meanwhile, referring to the literatures, we found that redissolveammonium hydroxide increasingly could increase the recovery rate of polypeptide group.Thought a series of experiment, we finally determined that the extraction method forpolypeptide group is the denaturized precipitation of mixed solvent (acetonitrile/acetone/water=50/30/20) in4times of volume, with the denaturized time of30mins and the roomtemperature as denaturized temperature.Sequence the polypeptide in the sample via electrospray quadrupole flight timetandem mass spectrum, compare the polypeptide sequence of the H group and that ofLC1+2+3group. Analyze the searched peptide fragments using blast MS. Then it couldobtain the corresponding protein in the contained peptide chain. We searched852peptidefragments from the healthy control group,1125peptide fragments from LC1+2+3group;comparing with these two groups, we found that there are nearly80different peptidefragments, among which scorings of7peptide fragments were more than20. We concludethat these seven groups of protein may be the candidate serum marker of lung squamouscell carcinoma. The8corresponding proteins were metastasis-associated proteinMTA1/3、Matrix metalloproteinase-16precursor、Cytokerantin-19、haptoglobin isoform2preproprotein、 breast cancer type1susceptibility protein isoform4/3/2/1、growth/differentiation factor15precursor、Complement C3precursor. We speculate thatMatrix metalloproteinase-16precursor of proteins can become potential biomarkers of lungsquamous cell carcinoma.Conclusion:1. the extraction method for polypeptide group is the denaturizedprecipitation of mixed solvent (acetonitrile/acetone/water=50/30/20) in4times of volume.2. Matrix metalloproteinase-16precursor of proteins we found can becomepotential biomarkers of lung squamous cell carcinoma.