Anja Pfenninger

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 1: methodology)

Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M − H]− with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a “new” reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MSn capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal β1→3 GlcNAc or Gal β1→4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (part 2: application to isomeric mixtures)

A complex mixture of isomeric neutral oligosaccharides from pooled human milk was analyzed by nano-electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer (QIT-MS) in the negative ion mode. Since deprotonated molecules of neutral oligosaccharides follow distinct fragmentation rules, which have been elucidated by using model compounds (see [1]), spectra obtained from consecutive CID experiments allowed the differentiation of isomers out of this highly complex mixture. With this method new human milk oligosaccharides of previously unknown isomeric structures have been identified, e.g., the occurence of three isomeric fucosylated lacto-N-hexaoses could be determined precisely, which have not been described before: (1) Fuc (α1 → 2) Gal (β1 → 3) GlcNac (β1 → 3) Gal (β1 → 4) GlcNac (β1 → 3) Gal (β1 → 4) Glc, (2) Gal (β1 → 4) GlcNAc [(α1 → 3) Fuc] (β1 → 3) Gal (β1 → 4) GlcNac (β1 → 3) Gal (β1 → 4) Glc, (3) Gal (β1 → 4) GlcNAc (β1 → 3) Gal (β1 → 4) GlcNac [(α1 → 3) Fuc] (β1 → 3) Gal (β1 → 4) Glc.

Mechanisms in MALDI analysis: surface interaction or incorporation of analytes?

Abstract Since the early days of matrix-assisted laser desorption/ionization (MALDI), the definition of a unified theory on the MALDI process is a major challenge. The new results presented in this paper clearly show that the idea of a uniform MALDI mechanism that accounts for the spreading applications has to be given up. Based on different preparation protocols distinct differences in the desorption/ionization process for carbohydrates in contrast to peptides/proteins are elucidated. Although isolated/incorporated and cluster-desorbed peptides/proteins are effectively entrained and cooled within the expanding plume of matrix clusters, as shown by a low degree of metastable analyte-ion fragmentation, a laser desorption and gas-phase cationization mechanism can be confirmed as the dominant part in ionization for neutral oligosaccharides which can be initiated even for particulate analyte material or deposits onto a matrix surface. The previously presented “lucky survivor-model” on cluster desorption of preformed ions thus needs this extension.

Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk.

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.

Observation of noncovalent complexes using laser-induced liquid beam ionization/desorption

Abstract A novel mass spectrometric method for the detection and identification of noncovalent biological interactions is described. The dimeric forms of gramicidin D and a leucine zipper polypeptide can be observed intact in the gas phase using Laser Induced Liquid Beam Ionization/Desorption (LILBID). Gramicidin D diluted to a concentration of 5·10 −5 M in ethanol and 1-propanol shows a laser-intensity- and solvent-dependent distribution in the monomer/dimer ratio, consistent with solution dimerization constants. Mass spectra of the leucine zipper peptide in methanol and ethanol show a charge state distribution varying from singly to triply charged monomer, with the relative maximum depending on the solvent and the laser intensity. Dissociation of the dimeric associations of gramicidin D and the leucine zipper peptide can be induced by increasing the intensity of the exciting laser. The desorption conditions strongly influence the preservation of weak noncovalent associations.

Mass Spectrometric Investigations of Human Milk Oligosaccharides

Human milk oligosaccharides (OS) have been fractionated by gel permeation chromatography (GPC) and analyzed by mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS) and electrospray ionization (ESI) ion trap/MS were used. Using a large human milk pool, the MALDI-TOF/MS analysis of high-molecular-mass GPC fractions showed that complex, multiply fucosylated and/or sialylated OS are present over a larger mass range than described previously Acidic oligosaccharides could be detected from low-retention-time GPC fractions with masses up to 4000 Da, which has not been reported before.