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dc.contributor.authorJiang, Hui
dc.contributor.authorDesaire, Heather
dc.contributor.authorButnev, Vladimir Y.
dc.contributor.authorBousfield, George R.
dc.identifier.citationHui Jiang, Heather Desaire, Vladimir Y Butnev, George R Bousfield, Glycoprotein profiling by electrospray mass spectrometry, Journal of the American Society for Mass Spectrometry, Volume 15, Issue 5, May 2004, Pages 750-758, ISSN 1044-0305, (
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dc.description.abstractThis work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHα (ovine luteinizing hormone, α-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn56 site of oLHα. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn56 site of oLHα was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition. Structural analysis of glycoproteins is a complicated task due to the carbohydrate's complex heterogeneity. Because of the complexity of the samples and the typically small amounts of material available, mass spectrometry is one technique that could provide structural information in most cases [1]. There are two common procedures for characterization of carbohydrates on glycoproteins by mass spectrometry. The first involves release of glycans, purification, and MS analysis 2 and 3. Although removing the protein from the carbohydrates could result in simpler mass spectra, information about which carbohydrates came from which site is lost. In addition, the proton-affinity of carbohydrates is weak. Their mass spectral signal to noise ratio is low, and quite often derivatization of the samples is necessary to increase ion signal [4]. An alternate approach to analyzing the carbohydrates on glycoproteins involves digestion of the glycoprotein with endoproteases and characterizing the resulting glycopeptides 5, 6, 7 and 8. The resultant glycopeptides may be analyzed by mass spectrometry with minimal clean-up. Peptide backbones generally have higher ionization efficiency than carbohydrates, producing more intense mass spectral signals and making accurate mass measurement easier. In addition, proteolytic fragments on either side of the amino acid carrying the carbohydrate can be used to reveal the specific site from which the carbohydrate originated [9]. When multiple glycosylation sites are present on a single glycoprotein, site-specific (glycopeptide) analysis is frequently require in order to determine which carbohydrates originated at which glycosylation site. Recently, Lebrilla and co-workers have demonstrated an effective technique for site-specific glycopeptide analysis using a nonselective enzyme and MALDI-FT MS [10]. To further develop this approach as an effective analytical technique, we compare herein various methods of site-specific glycopeptide analysis using electrospray mass spectrometry. Several different ESI-MS strategies for detection and composition determination of glycopeptides have been previously demonstrated. For example, parent ion scanning for the characteristic glycopeptide marker ions has been carried out 1 and 11. Alternatively, direct determination of glycopeptide composition has been achieved by accurate mass measurements on a qTOF mass spectrometer for various glycoproteins 7 and 8. Quadrupole ion traps have also been used in the analysis of glycopeptides 12 and 13. The focus of this study is to profile the composition of the glycoforms present at a specific site on the protein, and compare several previously proposed ESI-MS techniques in order to determine the most effective strategy for glycopeptide analysis on electrospray mass spectrometers. The ESI-MS techniques to be compared include composition profiling using MS data on a quadrupole ion trap, quadrupole time of flight, and triple quadrupole mass spectrometer. These studies are compared with results achieved via neutral loss scanning or precursor ion scanning experiments on a triple quadrupole mass spectrometer. The glycoprotein chosen for these studies is luteinizing hormone (LH). This glycoprotein is one of three structurally related glycoprotein hormones involved in the regulation of reproduction. It consists of two noncovalently bound α- and β-subunits. Both the common α- and hormone-specific β-subunits are glycosylated. The glycosylation sites on both of these subunits play important roles in the structure and the function of the protein. Oligosaccharides attached to the human α-subunit at Asn78 are important for proper folding of the protein [14]. In addition, the type of oligosaccharides attached to a second glycosylation site (Asn52) have been shown to influence receptor-binding affinity and are essential for full expression of biological activity at the cellular level [15]. (This site is numbered Asn56 in ovine and other mammalian α-subunits.) Glycosylation on the β-subunit has also been demonstrated to have biologically significant consequences on circulatory clearance and signal transduction [16]. Since each of the glycosylation sites have been implicated in different biological roles, it would be useful to know which carbohydrates are present at each site on the protein. By subjecting this protein to the mass spectrometric experiments herein, site-specific glycosylation information is obtained.en_US
dc.relation.ispartofseriesJournal of the American Society for Mass Spectrometry;
dc.relation.ispartofseries;V.15, No.5
dc.titleGlycoprotein profiling by electrospray mass spectrometryen_US

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