Microcolumn separations and the analysis of single cells.

Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.

Quantitative amino acid analysis of subnanogram levels of protein by open tubular liquid chromatography.

A method is described for the hydrolysis and quantitative amino acid analysis of as little as 0.1 ng (4 fmol) of protein. Hydrolysis is performed by using a gas-phase method. The resulting amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. To our knowledge this is currently the lowest level of protein to be quantitatively analyzed, as well as the smallest volume in which derivatization has been accomplished. It was possible to quantitatively determine 14 amino acids with an error of 5.0% for known protein and 8.5% for a protein whose identity was unknown to the researcher.

The carbohydrate components of hydrolysates of gastric secretion and extracts from mucous glands of the gastric body mucosa and antrum.

1. The sugars and amino sugars of hydrolysates of gastric secretion were determined by gas-liquid chromatography. 2. All the gastric aspirations examined showed on hydrolysis the presence of fucose, galactose, mannose, glucose, galactosamine, glucosamine, N-acetylneuraminic acid and sulphate. 3. Galactose and glucosamine were always found in equimolar amounts, but the galactose/galactosamine ratio in different aspirations was 2:1, 3:1, 4:1 or 5:1. Repeated gastric aspirations of each subject examined showed constant ratios of these carbohydrate components. 4. Fucose and sialic acid appear to be related to glucosamine and galactosamine respectively. 5. The carbohydrate components of extracts from the mucous glands of the body mucosa and antrum did not differ from those of gastric secretion.

A comparative study of the major glycoprotein isolated from normal and neoplastic gastric mucosa.

The isolation and composition of glycoproteins from mucosae of normal stomachs, of stomachs with gastric ulcer, and of stomachs with carcinoma is described. The glycoproteins from the mucosae of normal stomachs and with gastric ulcer showed virtually the same carbohydrate and amino acid content as the principal gastric glycoprotein isolated from gastric aspirates. They all revealed a common basic carbohydrate composition: galactose, fucose, glucosamine, and galactosamine were present in approximate molar ratios of 4:3:3:1. THE RESULTS SUGGEST THAT THE GLYCOPROTEINS ISOLATED FROM GASTRIC ASPIRATES FROM NORMAL AND NEOPLASTIC GASTRIC MUCOSAE SHARE A NUMBER OF STRUCTURAL FEATURES: (1) a protein core with a characteristic amino acid composition; (2) the range of sugars forming the carbohydrate side chains; (3) galactosamine approximately equimolar with the sum of threonine and serine; (4) galactose approximately equimolar with the sum of glucosamine and galactosamine; (5) absence of mannose; (6) a high carbohydrate content (80-85%); and (7) blood group activity. The neoplastic glycoproteins differed from the normal glycoproteins in that the quantitative relationships of the carbohydrate components of the neoplastic glycoproteins showed variations dividing the extracts investigated into groups, each group with a distinctive and constant carbohydrate composition. The blood group specificity of 15 out of 24 cases investigated differed from that of the hosts' red cells.

The isolation and composition of the major glycoprotein from human gastric aspirates.

The isolation of the principal glycoprotein from human gastric aspirates and the determination of its carbohydrate and amino acid composition is described.Ninety-nine individual gastric aspirates were investigated. Eighty-two were eluted on Bio-gel P150 and the carbohydrate and amino acid composition of each non-retarded fraction was determined. Fifteen of these non-retarded fractions were chromatographed again on Ecteola cellulose. Seventeen aspirates were precipitated with cetylpyridinium chloride. The carbohydrate, amino acid, and sulphate contents of the subfractions eluted on Ecteola cellulose and cetylpyridinium chloride precipitates were determined. The results suggest that the non-retarded fractions are composed of glycoproteins with a constant basic composition but polydisperse with respect to the sulphate contents and the terminal sugar residues which are associated with blood group specificity. It was found possible to correlate and identify in chemical terms the blood group specificity of all the glycoproteins investigated. No significant differences were detected between the carbohydrate and amino acid composition of each of the non-retarded fractions, the subfractions eluted on Ecteola cellulose, and the cetylpyridinium chloride precipitates. The sulphate content of the isolated glycoproteins was found to vary between zero and a sulphate: glucosamine ratio of 2:3. The subfractions of Ecteola cellulose showed that 20-50% of the glycoproteins were sulphated. The data also suggest that the isolated glycoprotein is the principal carbohydrate-containing fraction of the gastric secretion as it contained 68-96% of the total carbohydrate content of the gastric aspirates investigated.

Determination of naphthalene-2,3-dicarboxaldehyde-labeled amino acids by open tubular liquid chromatography with electrochemical detection.

Naphthalene-2,3-dicarboxaldehyde (NDA) has been investigated as a new derivatizing reagent for the electrochemical detection of tagged amino acids. Gradient elution allowed for the separation of 18 NDA-derivatized amino acids on an open tubular liquid chromatography column in less than 50 min. Gradient elution and electrochemical detection were found to be compatible. A detection limit of 36 amol was obtained for the asparagine-NDA derivative. The usefulness of this technique for quantitation was demonstrated by the analysis of the NDA-tagged hydrolysis products from bovine chymotrypsinogen.

Quantitative amino acid analysis of individual snail neurons by open tubular liquid chromatography.

A method is described for the determination of amino acids in individual cells. The amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. It was possible to quantitatively determine 17 amino acids in three different neurons of the land snail Helix aspersa. Quantitation was accomplished through the use of two internal standards and a calibration curve. Alanine was found to be the most abundant amino acid by about a factor of 2 over glutamine in all three types of neurons. This method has the advantages of sensitivity in the attomole range (5 X 10(-9) M) and selectivity for a specific class of compounds and is at least as reliable as other methods used for single-cell analysis.