ABSTRACT
This paper reports the use of a quadrupole time-of-flight (Q-TOF) mass spectrometer fitted with a matrix-assisted laser desorption/ionization (MALDI) ion source for the analysis of neutral and acidic glycosphingolipids. All compounds gave strong [M + Na]+ ions with 2,5-dihydroxybenzoic acid as the matrix, with no loss of sensitivity with increasing mass as was observed from the corresponding ions produced by electrospray. Neutral glycosphingolipids showed negligible in-source fragmentation but sialylated compounds fragmented by loss of sialic acid. However, these losses were not accompanied by unfocused post-source-decay ions as observed with MALDI-reflectron-TOF instruments. The MS/MS spectra were almost identical to those obtained by electrospray. Fragmentation of all compounds was mainly by glycosidic cleavage to give ions, both with and without the ceramide moiety, which defined the carbohydrate chain sequence. Weak ions which defined the sphingosine chain length and abundant ions, produced by loss of the acyl chain, were present when this chain contained a 2-hydroxy group. The technique was applied to the identification of ceramide-trihexosides present in tissues from mice genetically modified to model one of the glycolipid storage diseases (Fabry disease).
Subject(s)
Glycosphingolipids/chemistry , Animals , Brain Chemistry , Carbohydrate Sequence , Cattle , Gaucher Disease/metabolism , Humans , Kidney/chemistry , Liver/chemistry , Mice , Molecular Sequence Data , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spleen/chemistryABSTRACT
N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.
Subject(s)
Glycoproteins/chemistry , Oligosaccharides/analysis , Sequence Analysis/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Carbohydrate Sequence , Molecular Sequence DataABSTRACT
Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.
Subject(s)
Genes, Tumor Suppressor , Neoplasm Metastasis/genetics , Proteins/genetics , Receptors, Neuropeptide/analysis , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , GTP-Binding Proteins/physiology , Humans , Kisspeptins , Ligands , Molecular Sequence Data , Neoplasm Metastasis/prevention & control , Proteins/isolation & purification , Proteins/metabolism , Rats , Receptors, G-Protein-Coupled , Receptors, Kisspeptin-1 , Receptors, Neuropeptide/metabolism , Tumor Suppressor ProteinsABSTRACT
This paper reports the use of an experimental matrix-assisted laser desorption/ionisation (MALDI) ion source fitted to a quadrupole time-of-flight (Q-Tof) mass spectrometer for the analysis of carbohydrates, particularly the N-linked glycans from glycoproteins. Earlier work on the Q-Tof instrument, using electrospray ionisation, gave excellent MS/MS spectra, particularly from the [M + Na]+ ions, but suffered from the major disadvantages that the signal was often split between singly and multiply charged ions and that sensitivity fell dramatically as the molecular weight of the carbohydrate rose. The MALDI ion source did not suffer from these problems and the instrument produced excellent MS and MS/MS spectra from small amounts of complex, underivatised glycans as well as those derivatised at the reducing terminus. Positive ion MS spectra of sialylated glycans recorded on the new instrument were much less complex than those recorded with a conventional MALDI-TOF instrument because of the absence of ions resulting from metastable (post-source decay, (PSD)) fragmentations occurring in the flight tube. However, considerable fragmentation by loss of sialic acid still occurred. MS/MS spectra of the [M + Na]+ ions from all compounds were almost identical to those recorded earlier with the electrospray-Q-Tof combination and far superior to MALDI-PSD spectra recorded with reflectron-TOF instruments. Spectra are shown for neutral and sialylated N-linked glycans from chicken ovalbumin, riboflavin binding protein, alpha1-acid glycoprotein, bovine fetuin and ribonuclease B, both as free glycans and as those derivatised at their reducing termini. The technique was applied to the structural determination of N-linked glycans from human secretory IgA and Apo-B 100 from human low-density lipoprotein.