Interaction Between Annexin I And IgG In Esophageal Squamous Cell Carcinoma Development And Analysis Of Chinese Serum Proteome

ObjectiveEsophageal squamous cell carcinoma (ESCC) is a major kind of malignant tumor, and it is the fourth leading cause of cancer - related death in China, yet its molecular mechanisms are still poorly understood. Previous studies demonstrated that abnormal expression, function and translocation of annexin I are often found in several kinds of cancer, especially in ESCC. However, the molecular mechanisms remain elusive.Annexin I is a member of the family of annexins that are regarded as phos-pholipid and calcium - binding proteins. It may interact with hosts of proteins. Moreover, there are lots of potential phosphorylated sites in annexin I sequence, indicating that annexin I may play roles in cell signaling. Hence, changes of annexin Is biological behavior could result in the changes of related cell signaling and might further promote the development of cancer. Normal cell life needs the interactions between proteins, so exploring annexin I and its associated proteins interactions will help to understand the mechanisms of ESCC carcinogenesis.Materials and MethodsMaterials1. Human normal esophageal tissue and ESCC tissue2. Human ESCC cell line - EC01563. Immunoprecipitation related reagents4. Western blot related reagentsMethods1. Preparation of pooled normal esophageal tissue and ESCC specimen2. Extraction of tissue and cell line protein3. Immunoprecipitation of annexin I and IgGResults1. Interaction between annexin I and IgG in normal esophageal tissue2. Attenuated interaction between annexin I and IgG in ESCC3. The binding of IgG to annexin I in a calcium — dependent manner4. Endogenous IgG level is higher in normal esophageal tissue than ESCCDiscussionIn normal esophageal tissue, IgG may interact with annexin I in calcium -dependent manner, and the nature of such interaction might be non - covalent, but different from the recognition between antibody and antigen. In ESCC, interaction of annexin I and IgG becomes week, which could be due to: 1) The level of annexin I is lower in ESCC;2) The level of IgG is lower in ESCC;3) The concentration of calcium is lower in ESCC.ConclusionIn normal esophageal tissue, IgG may interact with annexin I in calcium -dependent manner. In ESCC, interaction of annexin I and IgG becomes week, which might be associated with the developmant of ESCC.ObjectiveSerum is an amorphous and important component of blood. Many researches have proved that quantitative and qualitative changes of serum proteins are related to physiological or pathological states of human body. With the development of proteomics, more and more proteomics techniques are used to profile proteins existing in serum and identify diseases related serum markers in order to approach early diagnosis and effective treatment of diseases.Historically, 2 - DE has been the primary method of separation and comparison for complex protein mixtures, and it has been used to analyze human serum. Despite its prominent advantages, e. g. high resolution and sensitivity, 2 — DE requires manual dexterity and precision to reproduce precisely and is thus not well - suited as a high - throughput technology. Recently multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples and be a complementary to 2 - DE based analysis. Compared with traditional proteomic 2 -DE, shotgun, serving as a powerful tool to separate and identify proteins from complex protein mixtures , possesses the virtues of high efficiency, time and labor saving.In this study, we conducted research on the protein components in sera of healthy Chinese using shotgun proteomics. The resulting non - redundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.Materials and MethodsMaterials1. Pooled male and female serum specimen2. Regents and equipments for removing high abundant serum proteins3. Regents and equipments for concentrating serum proteins4. Regents for digesting flow through and bound protein mixtures5. Regents and equipments for separating and analyzing the digestive pep-tides6. Regents and equipments for identifying digestive peptides Methods1. Preparation of pooled male and female serum specimen2. Depletion of the highly abundant serum proteins3. Protein concentration4. Trypsin digestion of flow through and bound protein mixtures5. RP - HPLC - MS/MS shotgun analysis6. Protein identification7. Bioinformatic analysisResults1. Data production of the shotgun MS identification2. Proteins in bound and unbound fractions3. Removal of redundancy in datasets4. Shotgun sequencing of serum proteins from healthy pooled male and female serum5. Function and localization analysis of pooled serum proteinsDiscussion1. Data quality and sensitivityIn this study, the serum donors were healthy Han - nationality Chinese. With the shotgun strategy of proteomics analysis, a total of 944 distinct proteins were identified. With multi - peptide identifications ( identified by more than one peptides) , a total of 3020 proteins were identified by HPPP participating laboratories , which were confirmed by more than one laboratory or more than one specimen in HPPP datasets. The 3020 proteins include 206 proteins identified by our group with at least two distinct peptides per protein. Additionally, using high confidence identifications (Xcorr ^1.9, 5:2.2, ^3.75, ACn^ 0. 1, Rsp^4.0-, fully triptic) with at least two distinct peptides per protein from the same experiment and specimen in the same laboratory, a total of 551 serum proteins were identified and considered to be high - quality proteins in HPPP data-sets, in which 185 proteins were found in our lists. And 86% (159/185) of the high - quality proteins have been confirmed by other PPP participating laboratories in HPPP datasets. Still, nearly 66% of our high - quality proteins are secreted proteins that are regarded as classical plasma proteins. It is of particular interest that we have also identified some low abundance proteins ( ng/ml) , which were known to be involved in disease processes. So we could draw a conclusion that our data are relative trustworthy.2. Parallel analysis of bound and unbound fractionsHerein, 351 proteins were only found in bound but not unbound fraction, most of which have only one peptide identified and might be some other proteins, which were also removed during the process of depletion in this study. That suggested that the depletion would remove some proteins except for the top - six captured ones. It is necessary to do the parallel analysis of bound and unbound fractions to obtain more global profile of the plasma proteome.ConclusionWe used shotgun strategy to investigate the serum proteome of Han - nationality Chinese. Total 944 non - redundant proteins were identified with stringent filter criteria. After compared our data with that of HPPP dataset under i-dentical filter parameters, great overlap ratio was found. The resulting non -re-dundant list of Chinese serum proteins might enrich the dataset of human plasma proteome and prove valuable clues to the disease markers discovery.