ovalbumin and 2-mercaptoacetate

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.

Topics: Amino Acids; Animals; Hydrochloric Acid; Hydrolysis; Lactoglobulins; Ovalbumin; Proteins; Sensitivity and Specificity; Serum Albumin, Bovine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioglycolates

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2-4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophage-enriched, Fc receptor-bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4 degrees C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.

Topics: Animals; Antimetabolites; Ascitic Fluid; Cell Count; Cell Survival; Centrifugation, Density Gradient; Edetic Acid; Egtazic Acid; Galactosidases; Galactosyltransferases; Glucuronidase; Inflammation; L-Lactate Dehydrogenase; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Ovalbumin; Salmonella enteritidis; Sialyltransferases; Thioglycolates; Time Factors