title: n-Alkyl p-aminobenzoates as derivatizing agents in the isolation, separation, and characterization of submicrogram quantities of oligosaccharides by liquid secondary ion mass spectrometry.
abstract: In this laboratory we are pursuing a comprehensive strategy for isolation and characterization of oligosaccharides from glycoproteins that are available only in limited quantities. To improve sensitivity in the analysis by liquid secondary ion mass spectrometry, we have investigated the relative behavior of a homologous series of n-alkyl esters of p-aminobenzoic acid as derivatizing agents. Ethyl p-aminobenzoate, the derivatizing agent used in many of our earlier studies, is one of these compounds. Our experiments using the hepatasaccharide maltoheptaose (M7) as a model oligosaccharide establish that by lengthening the alkyl chain from methyl to n-tetradecyl, a concomitant increase in the molecular ion abundance is obtained. The increase is a factor of 10 when 1 microgram of derivatized M7 is analyzed, and as much as 40 when 0.1 microgram of sample is examined. This series of derivatives of maltoheptaose form a suite of relatively abundant fragment ions in the negative ion mode as expected from our previous studies with the ethyl ester. Although very high mass spectral sensitivities were achieved with M7 n-tetradecyl and n-decyl p-aminobenzoates, the yields of derivative obtained were significantly lower than those obtained for M7 n-octyl, n-hexyl, n-butyl, ethyl, and methyl p-aminobenzoates, despite improvements made in the derivatization procedure. When analyzing biological samples, n-octyl and n-hexyl p-aminobenzoate were found to be optimal considering both yield of derivative and mass spectral sensitivity. This improved method of derivatization was incorporated into a simple but effective procedure for dealing with very small quantities of heterogeneous samples of oligosaccharides, such as those released from 250 micrograms (1 nmol) of nicotinic acetylcholine receptor from Torpedo californica and 90 micrograms (2 nmol) of human alpha 1 acid glycoprotein.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
authors.forename: L
authors.initials: L
authors.lastname: Poulter
authors.forename: R
authors.initials: R
authors.lastname: Karrer
authors.forename: A L
authors.initials: AL
authors.lastname: Burlingame
mesh_terms: D010129:4-Aminobenzoic Acid / Q000138:chemical synthesis; D002240:Carbohydrate Sequence; D005936:Glucans / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents / Q000138:chemical synthesis; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D009961:Orosomucoid / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry; D062366:para-Aminobenzoates
chemical_list: D005936:Glucans; D007202:Indicators and Reagents; D009844:Oligosaccharides; D009961:Orosomucoid; D011978:Receptors, Nicotinic; D062366:para-Aminobenzoates; C031146:maltoheptaose; D010129:4-Aminobenzoic Acid