ABSTRACT
Proteomic analysis combining two-dimentional electrophoresis (2DE) and mass spectrometry (MS) has the potential for a wide range of applications in biological and medical sciences, as protein screening in tissues obtained from healthy and diseased conditions can determine drug targets and diagnostic markers. Conventionally, amniotic fluid (AF) samples are routinely used for prenatal diagnosis of a wide range of fetal abnormalities. Proteomics have already been applied in the analysis of tissues from fetuses with Down's syndrome, in order to detect differences in their protein profile as compared to the normal profiles and to determine possible diagnostic tools. A detailed protein 2DE for the normal human AF has not been reported. In the present study, the 2D protein database of the normal human AF supernatant (AFS) was constructed. Ten AFS samples from women carrying normal fetuses were analysed by 2DE. A mean of 412 spots per gel were analyzed and protein identification was carried out by MALDI-MS and MALDI-MS-MS. A 2D protein map comprising of 136 different gene products was constructed. The majority of the identified proteins are regulatory proteins, enzymes, secreted proteins, carriers and immunoglobulins. Twelve hypothetical proteins were also included. The normal AFS proteome map is a valuable tool for the study of aberrant protein expression and the search for proteins as possible markers for the prediction of abnormal fetuses.
Subject(s)
Amniotic Fluid/chemistry , Pregnancy Proteins/analysis , Proteome/analysis , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Mass Spectrometry , Peptide Mapping , Pregnancy , Prenatal Diagnosis , Proteomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The human immature T-cell line CCRF-CEM is widely used for all kinds of in vitro studies in biochemistry, biology, toxicology and medicine. Knowledge about protein expression is limited and no comprehensive study on the proteome of this cell type has been reported to date. Proteomics technologies were applied in order to analyse the proteins of the CEM cell line. The proteins were separated by two-dimensional (2-D) gel electrophoresis and analysed by MALDI-MS and MALDI-MS-MS following in-gel digestion with trypsin and, finally, protein identification was carried out by peptide mass fingerprint (PMF) and post source decay (PSD), respectively. Approximately 4,500 spots, excised from four 2-D gels, were analysed. The analysis resulted in the identification of about 1,150 proteins, the products of 451 different genes. The majority of the identified proteins were enzymes, regulatory proteins and transporters, while leukocyte markers and oncogenes were also included. The CCRF-CEM cell database today represents one of the largest 2-D databases for eukaryotic proteomes, forming the basis for future expressional studies at the protein level.
