Coupling of fully automated chip electrospray to Fourier transform ion cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing.
ABSTRACT The NanoMate robot has been coupled to a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer at 9.4 T and implemented for the first time for complex carbohydrate analysis. It was optimized in the negative ion mode to achieve automated sample delivery on the chip along with increased sensitivity, ultra-high resolution and accurate mass determination. A novel bracket has been designed to allow a reliable mounting of the NanoMate to the Apollo electrospray ionization (ESI) source of an APEX II instrument. The notably higher efficiency of ionization for compositional mapping of complex mixtures and feasibility for fragmentation analysis of components by sustained off-resonance irradiation collision-induced tandem mass spectrometry (SORI-CID MS2) has been demonstrated on a glycoconjugate mixture containing O-glycosylated sialylated peptides from urine of a patient suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease), previously analyzed by capillary-based nanoESI-FTICRMS, and of a healthy control person. Due to its potential to generate highly charged ionic species, reduce the in-source fragmentation, increase sensitivity, reproducibility and ionization efficiency, along with the ability to generate a sustained and constant electrospray, this method can be considered as a new platform for advanced glycomics.
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ABSTRACT: IntroductionA novel approach of ion mobility tandem mass spectrometry (IMS-MS/MS) is applied to analysis of human glycourinome to obtain carbohydrate pattern data of congenital disorders of glycosylation patient. Overlapping of the complex carbohydrate mass range landscape has been highly reduced upon IMS-MS procedure, allowing more efficient identification by mapping and sequencing of glycan precursor ions, following their separation by mobility, according to difference in drift time through the traveling wave IMS cell. Intact and truncated N- and O-glycan structures modified by sialylation and fucosylation were identified according to their drift time separated molecular ions and submitted to fragmentation in a narrow mass window. IMS CID MS/MS AnalysisThe fragmentation spectra generated from the IMS separated precursor ions contain series of fragment ions maintaining the same mobility as their parent ions, and the assignment accuracy can be significantly enhanced. ConclusionAccording to the specific fragment ion patterns, carbohydrate epitopes described to be involved in pathological processes were assigned. A high potential of this glycomics-based strategy for clinical applications can be presented.Clinical Proteomics 06/2008; 4(1):47-57. DOI:10.1007/s12014-008-9010-3
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ABSTRACT: This review summarizes the methods, mainly based on mass spectrometry, for the structural determination of N- and O-linked carbohydrates that are post-translationally attached to a large number of proteins and which play a key role in determining the function and biophysical properties of these compounds. Analysis of these carbohydrates has proved difficult in the past due to their structural complexity. However, modern analytical methods such as mass spectrometry have the ability to elucidate most structural details at the concentration levels required for proteomics. This review describes methods for direct examination of glycoproteins by mass spectrometry, the release of N- and O-linked glycans from glycoproteins separated in sodium dodecyl sulfate polyacrylamide electrophoresis gels, and the analysis of these compounds by techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry provides the most rapid method for comparing glycan profiles and is probably most appropriate for clinical studies. One of the most promising techniques for determining the structures of N-glycans in proteomic studies is negative ion fragmentation of electrosprayed ions. This technique combines high throughput with ease of structural interpretation and provides structural details that are difficult to obtain by classical methods.Expert Review of Proteomics 02/2005; 2(1):87-101. DOI:10.1586/147894126.96.36.199 · 3.54 Impact Factor
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ABSTRACT: Cell surface and extracellular proteins are O-glycosylated, where the most abundant type of O-glycosylation in proteins is the GalNAc attachment to serine (Ser) or threonine (Thr) in the protein chain by an a-glycosidic linkage. Most eukaryotic nuclear and cytoplasmic proteins modified by a-linked O-GlcNAc to Ser or Thr exhibit reciprocal O-GlcNAc glycosylation and phosphorylation during the cell cycle, cell stimulation, and/or cell growth. Less-investigated types of O-glycosylation are O-fucosylation, O-mannosylation, and O-glucosylation, but they are functionally of high relevance for early stages of development and for vital physiological functions of proteins. Glycosaminoglycans are a-linked to proteoglycans via a xylose-containing tetrasaccharide, represented by linear chains of repetitive disaccharides modified by carboxylates and O- or/and N-linked sulfates. Analysis of O-glycosylation by mass spectrometry (MS) is a complex task due to the high structural diversity of glycan and protein factors. The parameters in structural analysis of O-glycans include determination of (i) O-glycosylation attachment sites in the protein sequence, (ii) the type of attached monosaccharide moiety, (iii) a core type in the case of GalNAc O-glycosylation, (iv) the type and size of the oligosaccharide portion, (v) carbohydrate branching patterns, (vi) the site of monosaccharide glycosidic linkages, (vii) the anomericity of glycosidic linkages, and (viii) covalent modifications of the sugar backbone chains by carbohydrate- and noncarbohydrate-type of substitutents. Classical and novel analytical strategies for identification and sequencing of O-glycans by MS are described. These include methods to analyze O-glycans after total or partial release from the parent protein by chemical or enzymatic approach or to analyze O-glycosylated peptides by mapping and sequencing from proteolytic mixtures. A recombination process of multiply charged glycopeptides with electrons by electron capture dissociation Fourier transform ion cyclotrone resonance (FTICR)-MS has been introduced and is instrumental for nonergodic polypeptide backbone cleavages without losses of labile glycan substituents. A method for O-glycoscreening under increased sensitivity and efficient sequencing as a combination of an on-line coupling of capillary electrophoresis separation, as well as an automated MS-tandem MS (MS/MS) switching under variable energy conditions collision-induced dissociation (CID) protocol, is beneficial for determination of O-acetylation and oversulfation (Bindila et al., 2004a; Zamfir et al., 2004a). O-glycomics by robotized chip-electrospray/ionization (ESI)-MS and MS/MS on the quadrupole time-of-flight (QTOF) and FTICR analyzers, accurate mass determination, and software for assignment of fragmentation spectra represent essentials for high-throughput (HTP) in serial screenings (Bindila et al., 2004b; Froesch et al., 2004; Vakhrushev et al., 2005). Dimerization of intact O-glycosylated proteins can be investigated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS after blotting.Methods in Enzymology 02/2005; 405:139-71. DOI:10.1016/S0076-6879(05)05007-X · 2.19 Impact Factor