scholarly journals A structural analysis of heparin‒like glycosaminoglycans using MALDI‒TOF mass spectrometry

2004 ◽  
Vol 18 (2) ◽  
pp. 185-201 ◽  
Author(s):  
Warren C. Kett ◽  
Deirdre R. Coombe
Keyword(s):  
Mass Spectrometry ◽  
Structural Analysis ◽  
Therapeutic Potential ◽  
Structural Data ◽  
Enzymatic Digestion ◽  
Maldi Ms ◽  
Sequence Information ◽  
Sulfated Oligosaccharides ◽  
Flight Mass Spectrometry ◽  
Basic Peptides

Mass spectrometry (MS) techniques have spear‒headed the field of proteomics. Recently, MS has been used to structurally analyse carbohydrates. The heparin/heparan sulfate‒like glycosaminoglycans (HLGAGs) present a special set of difficulties for structural analysis because they are highly sulfated and heterogeneous. We have used a matrix‒assisted laser desorption/ionization time of flight mass spectrometry (MALDI‒MS) technique in which heparin fragments are non‒covalently bound to basic peptides of a known mass, so as to limit in‒source desulfation and hence afford an accurate mass. We examined a range of different sized fragments with varying degrees of sulfation. The potential of combining the MALDI‒MS technique with enzymatic digestion to obtain saccharide sequence information on heparin fragments was explored. A disaccharide analysis greatly assists in determining a sequence from MALDI‒MS data. Enzymatic digestion followed by MALDI‒MS allows structural data on heparin fragments too large for direct MALDI‒MS to be obtained. We demonstrate that synthetic sulfated oligosaccharides can also be analysed by MALDI‒MS. There are advantages and limitations with this methodology, but until superior MS techniques become readily accessible to biomedical scientists the MALDI‒MS method provides a means to structurally analyse HLGAG fragments that have therapeutic potential because of their ability to bind to and functionally regulate a host of clinically important proteins.

Identification of Phosphorylcholine-Substituted Peptides by Their Characteristic Mass Spectrometric Fragmentation

2005 ◽  
Vol 11 (3) ◽  
pp. 335-344 ◽  
Author(s):  
Julia Grabitzki ◽  
Volker Sauerland ◽  
Rudolf Geyer ◽  
Günter Lochnit
Keyword(s):  
Mass Spectrometry ◽  
Ion Trap ◽  
Neutral Loss ◽  
Structural Data ◽  
Mass Spectrometric ◽  
Peptide Backbone ◽  
Fragmentation Behavior ◽  
Flight Mass Spectrometry ◽  
Characteristic Fragmentation ◽  
Fragmentation Patterns

Phosphorylcholine (PC)-substituted biomolecules are wide-spread, highly relevant antigens of parasites, since this small hapten has been found to be a potent immunomodulatory component which allows the establishment of long lasting infections of the host. Structural data, especially of protein bound PC-substituents, are still rare due to the observation that mass spectrometric analyses are mostly hampered by this zwitterionic substituent resulting in low sensitivities and unusual but characteristic fragmentation patterns. Here, we investigated the fragmentation behavior of synthetic PC-substituted peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization ion-trap mass spectrometry. We could show that the predominant neutral loss of a trimethylamine unit (Hoffmann elimination) leads to cyclic phosphate derivatives which prevent further fragmentation of the peptide backbone by stabilizing the positive charge at this particular side chain. Knowledge of this PC-specific fragmentation might help to identify PC-substituted biomolecules and facilitate their structural analysis.

scholarly journals Characterisation of the covalent structure of proteins from biological material by MALDI mass spectrometry ‣ possibilities and limitations

1998 ◽  
Vol 14 (1) ◽  
pp. 1-27 ◽  
Author(s):  
Martin Kussmann ◽  
Peter Roepstorff
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Biological Material ◽  
Protein Nmr ◽  
Structural Data ◽  
Maldi Ms ◽  
Protein Chemistry ◽  
Nmr Studies ◽  
Covalent Structure ◽  
Structure Of Proteins

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) has become a primary tool for the detailed characterisation of the covalent structure of proteins isolated from biological material, mainly because of its following potentials: high sensitivity and specificity, speed of analysis, appropriateness for mixture analysis, high tolerance towards contaminants, and compatibility with separation techniques, e.g., gel electrophoresis. These characteristics enable the structural analysis of proteins even if they are only available in limited amounts and/or in mixtures, and even if the protein preparations contain large amounts of salts, buffers, detergents and denaturants. Additionally, structural data can be generated within a relatively short time.Whereas X-ray crystallography and multidimensional NMR techniques can provide “absolute” structural data, i.e., a three-dimensional “picture” of the protein of interest, MALDI-MS-especially in combination with selective protein chemistry – yields information on particular aspects of the entire protein structure, e.g., primary structure, active site(s), binding sites, and posttranslational modifications, all of which are often of crucial interest for the understanding of the protein function. Taking into account that protein crystallography and protein NMR studies require large quantities of highly purified sample, MALDI-MS can be even more regarded as a powerful complement in protein structure analysis.This review aims at describing the state-of-the-art of MALDI-MS for characterisation of proteins from biological material by evaluating its potential and limitations.

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