食管鳞癌肿瘤相关抗原磷酸甘油酸激酶1的鉴定

Phosphoglycerate kinase 1 as a candidate of tumor-associated antigen identified from esophageal squamous cell carcinoma

目的:鉴定新的食管鳞癌肿瘤相关抗原.方法:食管癌EC0156细胞总蛋白先经亚组分预分离,有效富集胞质、胞膜和胞核等组分蛋白;胞质组分蛋白经SDS-PAGE分离后分别与食管癌患者血清或健康志愿者血清共孵育,分离血清结合蛋白条带;胶内酶解阳性蛋白条带,肽段经色谱分离后用SynaptTM HDMS质谱鉴定.候选蛋白进一步Western blot经免疫组化验证.结果:总蛋白经亚组分分离后,不同组分蛋白均得到有效富集.食管癌患者血清能与EC0156细胞胞质蛋白结合,即选择性识别肿瘤相关抗原.其中,43kDa蛋白条带与食管癌血清(41.4%,12/29)和对照(3.6%,1/28)结合阳性率具有明显差异.从该蛋白条带中共鉴定到磷酸甘油酸激酶、β-actin、蛋白酶体26s亚基、S-腺苷高半胱氨酸水解酶和磷酸核糖酰氨基咪唑羧化酶5个高可信度蛋白.磷酸甘油酸激酶(PGK1)定位于胞质和胞核,在食管癌组织中高表达(69.23%,18/26).结论:改良血清蛋白质组分析策略(mSERPA)可有效分离鉴定肿瘤相关抗原.PGK1是食管癌候选肿瘤相关抗原,在食管癌发生发展中可能发挥重要作用.

AIM： To investigate and identify novel tumor- associated antigens in esophageal squamous cell carcinoma （ESCC）. METHODS： Modified serological proteome analysis （mSERPA） strategy was used to separate and identify the candidate proteins. The subcellular protein fractions （cytosolic, membrane and nuclear fractions） of ESCC cell lines and EC0156 cells were extracted first and then cytosolic proteins were separated using SDS- PAGE. The separated proteins were incubated with different serum of ESCC patients （29 cases） or healthy controls （28 cases） respectively, and then one of the positive bands in 43 kDa was excised followed by in-gel tryptic digestion. Separated peptides were identified using a high definition mass spectrometry （HDMS）. Western blot and immunohistochemical staining （IHC） were used to validate possible candidates. RESULTS： Successful compartmental protein extraction was demonstrated by specific organelle markers. Serum samples of ESCC patients bound EC0156 cytoplasmic protein, suggesting selective recognition of tumor-associated antigen. 43 kDa protein band showed significantly higher positive binding rate with serum of ESCC patients （41.4%, 12/29） than with serum of healthy individuals （3.6%, 1/28）. Five high-confidence proteins were identified from the 43 kDa band using HDMS including phosphoglycerate kinase 1 （PGK1）, β-actin, proteasome 26S subunit, S-adenosylho- mocysteine hydrolase and hosphoribosylamino- imidazole carboxylase. Immunohistochemistry. Western blot analysis showed that PGK1 was located in both cytoplasm and nucleus, and had a higher expression in cancer tissues （69.23%, 18/26） than in normal esophageal epithelia. CONCLUSION： The mSERPA strategy is useful for tumor-associated antigen identification. As a new candidate of tumor-associated antigen, PGK1 was over-expressed in ESCC which may play a role in tumorigenesis of ESCC.