Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS
thesisposted on 22.05.2021, 10:55 by Zhuo Zhen Chen
Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.