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1093/glycob/cwj067 Advance Access publication on December 8, 2005
Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis
Willy Morelle2, Christophe Flahaut3, Jean-Claude Michalski2, Alexandre Louvet4, Philippe Mathurin4, and André Klein1,2,5
2 Unité Mixte de Recherche CNRS/USTL 8576, Glycobiologie Structurale et Fonctionnelle, IFR 118, Université des Sciences et Technologies de Lille 1, 59655 Villeneuve d’Ascq, France; 3Laboratoire de physiopathologie de la barrière hémato-encéphalique, E.A. 2465, IMPRT-IFR 114 Université d’Artois, rue Souvraz, SP18, 62307 Lens Cedex, France; 4Services d’Hépato-Gastroentérologie, Hôpital Huriez, CHRU Lille, France; and 5 Laboratoire de Biochimie et de Biologie Moléculaire, UAM de glycopathologies, Hôpital Calmette, CHRU Lille, Bld du Professeur Jules Leclerc, Lille 59037 Cedex, France
Introduction Changes in glycosylation associated with human diseases represent a rapidly growing field. Part of these diseases, directly related to a congenital defect, is termed congenital disorders of glycosylation (CDG). Since 1980, when Jaeken et al. (1980) described a new neurologic disorder, 17 different defective proteins have been shown to be involved in an N-glycan biosynthesis defect. These defects are classified into CDG Type I if there is a decrease in the biosynthesis or the transfer of the lipid-linked oligosaccharide precursor, dolichol-linked Glc3Man9GlcNAc2, or classified into CDG Type II when the defect occurs after the transfer to the glycoprotein (Grünewald et al., 2002). Other congenital defects can be included in this group of diseases, as defects of N-glycosylation such as I-cell disease, congenital dyserythropoietic anemia type II (hereditary erythroblastic multinuclearity with positive acidified-serum lysis test [HEMPAS]), congenital galactosemia, and congenital intolerance to fructose. Infants suffering from CDG usually have a severe phenotype with mental retardation, failure to thrive, gastrointestinal problems, and dysmorphia. Clinical presentations are extremely broad, with or without neurological symptoms. Therefore, CDG should be suspected in any multi-systemic disorders. Isoelectrofocusing of serum transferrin is the first step for the diagnostic of N-glycosylation defects, and this test is the most efficient diagnosis method available for the identification of these diseases. This assay is based on the number of sialic acid residues of the different transferrin glycoforms and reflects the absence of terminal sialic acids, variations in N-glycan branching or the complete absence of N-glycan on either one glycosylation transferrin site or both of these sites. The availability of this test in most clinical laboratories has made it possible to diagnose more than 500 patients suffering from CDG, even if the screening of this group of diseases is not generalized. It has also provided the opportunity of pointing out Nglycosylation variations in other congenital diseases such as galactosemia (Charlwood et al., 1998), hereditary fructose intolerance (Adamowicz and Pronicka, 1996), and cystic fibrosis (Larsson et al., 1998). This test is also used to demonstrate glycosylation alterations in serum glycoproteins in case of chronic alcohol intake. This is the only iatrogenic etiology resulting in acquired modification of glycosylation, and the alteration mechanism is poorly understood (Flahaut et al., 2003). Acquired glycosylation changes in human diseases also represent an extremely large field of study (Varki, 1999). N-glycosylation modifications in human serum glycoproteins have mainly been described in liver, inflammatory diseases, and cancers. In the group of liver diseases, many
Received on May 6, 2005; revised on December 5, 2005; accepted on December 6, 2005
Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases. Key words: cirrhosis/glycosylation disorders/mass spectrometry/N-glycan/N-glycome
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we describe the use of this technique in cirrhosis. 2115. Total serum N-glycome is a noninvasive test.. the MALDITOF MS spectrum of the N-glycans from an healthy subject reveals the presence of major ions at m/z 2274 corresponding to an oligosaccharide with a chemical composition of NeuAcMe2Hex5HexNAc4. Modification of the immunoglobulin glycosylation has been demonstrated in rheumatoid arthritis (Parekh et al. As a first application. and high throughput assay which could be used in any clinical chemistry laboratory.. 1985) and in other autoimmune diseases (Basset et al. reproducible.. The MALDI-MS spectrum of N-glycans released from human serum of a patient with a decompensated cirrhosis is shown in Figure 1C. the N-glycans were enzymatically released with peptide N-glycosidase F (PNGase F). 1996). The ions at m/z 2944 correspond to a trisialylated oligosaccharide with a chemical composition of NeuAcMe3Hex6HexNAc5.. 2004). 2002). However.. relatively simple. As shown in Figure 1A. modifications in N-glycosylation have been demonstrated in hepatic cirrhosis (Anderson et al.. The sialic acids are stabilized by methylesterification of the carboxyl group. The monosialylated oligosaccharide is observed at m/z 1969. 1648. and (3) sialylated oligosaccharides at m/z 2318 and 2623 that were present as trace amounts in the healthy subject N-glycome are major components. which would represent diagnosis or prognosis markers of diseases. haptoglobin. with an increase of the relative intensities of ions at m/z 1486. Ryden et al. The MALDI-MS spectrum of the desialylated oligosaccharides (Figure 1D) confirms the previous observations. sensitive. Total serum N-glycome was analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) before and after chemical desialylation.. by linkage analysis and by electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Callewaert et al. Callewaert et al. However. reproducible. Variations of total serum N-glycome of two other patients The comparison of the MALDI-TOF MS spectra of the methylesterified and desialylated total serum N-glycome of patient Y (Figure 1E and F) and of patient X (Figure 1C and D) both with a decompensated cirrhosis and with acute alcoholic hepatitis (Table 2) indicates differences in the relative intensities of two groups of ions: (1) ions at m/z 1486 and 1648 are less intense and (2) ions with an m/z superior to 2900 are more intense in patient Y. Total serum N-glycome corresponds to the global qualitative study of all glycoprotein N-linked glycans present in the human serum and gives a general overview of the glycosylation of all serum glycoproteins that are essentially synthesized by the liver and by the immunoglobulin-secreting plasma cells. and sialylation of N-glycans on various serum glycoproteins. and α1-antitrypsin (Goodarzi and Turner. so that it may be used in a clinical chemistry laboratory... They have validated their protocol with the diagnoses of CDG (Callewaert et al. 1867. 2000). 2003) and established the importance of total serum N-glycome in monitoring the progression of chronic liver diseases (Callewaert et al. and 1851 represent ∼40% of the relative intensity of all the ions. 1993).. 1543.. and 2029 (Hex6HexNAc5) (Figure 1B). The chemical compositions of each ion in the spectrum are summarized in Table 1. have developed a protocol for profiling N-glycans on a DNA sequencer combined with a highthroughput multi-step method. 2003). 1689. Methylesterification of the carboxyl group is used to avoid the loss of terminal sialic acids during MALDI-TOF analysis and therefore allows simultaneous analysis of neutral and sialylated oligosaccharides in the positive mode (Powell and Harvey. 1989). 1810. Recently. such an assay needs to be of high throughput. none of these glycosylation modifications are used for diagnosis or prognosis. in hepatocellular carcinoma (Yamashita et al. In the second part of this study. These ions correspond to three groups of three oligosaccharides: trisialotriantennary Results After the denaturation of the proteins/glycoproteins present in human serum. 2002. 1705. in that blood sample can be obtained without inducing major trauma to the patient. and 2013. we describe an extremely simple method for the acquisition of total serum N-glycome–desialylated or not–on the basis of mass spectrometric approach of enzymatically released N-glycans purified by a single-step solidphase extraction. 1851. Any alterations in the liver and/or B-lymphocyte physiology might influence glycosylation mechanisms and show relative N-glycan differences. 1810 (Hex5HexNAc4DeoxyHex1). In this study. we elucidate the primary structure of N-glycans present in the total mix of serum glycoproteins in healthy donors and patients with cirrhosis. The discovery of new biomarkers is crucial for the diagnosis or prognosis of diseases. and 2420 corresponding to fucosylated oligosaccharides are more intense. and the corresponding fucosylated oligosaccharides are observed at m/z 2420 and 2115. 1648. The oligosaccharides were then purified in a single-step procedure by solid-phase extraction on porous graphitized carbon 282 . and the glycosylation changes observed are discussed. The reduction of sample heterogeneity by chemical desialylation of the N-glycans confirms this oligosaccharide distribution. The oligosaccharides were also characterized by MALDI-MS before and after exoglycosidase digestion. (2) ions at m/z 1810. 1502. 1988). MALDI-TOF analysis of N-linked glycans present in total human serum The released N-glycans were either chemically desialylated or methylesterified and analyzed by MALDI-TOF MS.. capable of isolating N-linked oligosaccharides (Papac et al. sensitive. 1998) have been characterized in chronic or acute inflammation. fucosylation.W. The spectrum presents three major differences with the MALDI-MS spectrum of the N-glycans of the healthy subject: (1) neutral oligosaccharide ions at m/z 1486. and easy to interpret. and the major reason is the non-availability of a simple. and the major ions are observed at m/z 1664 (Hex5HexNAc4). (PGC) columns. 2001). α1-acid glycoprotein (De Graaf et al. α1-antichymotrypsin (Hachulla et al. and in case of chronic alcohol intake (Flahaut et al. Modification of branching. 1998. Morelle et al.
2850. MALDI-TOF MS spectra of the methylesterified total serum N-glycome of patient Z (Figure 1G and H) with chronic hepatitis C and with fibrosis differ from the control serum N-glycome by an increase of ions at m/z 1486. 3090. the ions in each group corresponding to the non-. 3.and 6-linked galactose disappear and there is a concomitant increase of terminal galactose. mono-. representative of the biantennary complex glycans. with 2.6-linked mannose suggests that minor tri. (F) desialylated N-linked oligosaccharides from cirrhotic patient Y. oligosaccharides at m/z 2944. and 2285) together with an increase of fucosylated oligosaccharides as attested by the ions at m/z 2244. and 3212 almost absent in the normal N-glycome (Figure 2). Key features of the linkage analysis of the normal serum N-glycome are as follows. 1907. The distribution of the oligosaccharides was the same as the one observed previously and confirmed the important proportion of neutral oligosaccharides in the N-glycome of patients with decompensated cirrhosis (ions at m/z 1836. and a minor amount of terminal N-acetylglucosamine is 283 . Spectra obtained from (A) methylesterified N-linked oligosaccharides from normal subject. and tetrasialotetraantennary oligosaccharides at m/z 3616. trisialotetraantennary oligosaccharides at m/z 3309. 3455. Methylation analysis and linkage analysis of total human serum N-glycans The N-glycans of human serum glycoproteins were permethylated after chemical desialylation or not and analyzed by MALDI-TOF MS. and difucosylated compounds respectively.Total serum N-glycome in human diseases Fig. 1648. (D) desialylated N-linked oligosaccharides from cirrhotic patient X. The data are presented in Table 3. 2111. (G) methylesterified N-linked oligosaccharides from patient Z suffering from chronic hepatitis C. MALDI-TOF MS analysis of total human serum N-glycome.and/ or tetraantennary structures are present. 2081. 2489. 1866. The permethylated N-glycans and their desialylated counterparts were then hydrolyzed and analyzed using gas chromatography mass spectrometry (GC-MS) as their partially methylated alditol acetate derivatives. 2040. and 3906. and 2966 and the presence of oligosaccharide ions at m/z 2315. 2318. (1) Comparison of the relative abundance of the 2-linked mannose. (C) methylesterified N-linked oligosaccharides from cirrhotic patient X. indicating that sialic acid residues were mainly attached to the 6-position of galactose residues. (2) after desialylation. and 3236. (E) methylesterified N-linked oligosaccharides from cirrhotic patient Y. 1. and (H) desialylated N-linked oligosaccharides from patient Z. (B) desialylated N-linked oligosaccharides from normal subject. (3) most of the N-acetylglucosamines are 4-linked.4-linked mannose and 2. and 2623 (Figure 1A and G) confirmed by the increase of ions at m/z 2013 in the desialylated sample spectrum (Figure 1B and H). and 3601. 3760. 2605.
Morelle et al. Table I.W. Structure of N-linked glycans from total human serum N-glycome m/z A 1486 1502 1543 1648 1664 B 1836 1866 1907 2040 2070 C 929 944 965 1032 1047 Chemical composition Hex3HexNAc4DeoxyHex Hex4HexNAc4 Hex3HexNAc5 Hex4HexNAc5DeoxyHex Hex5HexNAc4 Structure 1689 2081 1052 Hex3HexNAc5DeoxyHex Hex4HexNAc5 Hex5HexNAc4DeoxyHex Hex4HexNAc5DeoxyHex Hex5HexNAc5 NeuAcMeHex5HexNAc4 1705 1810 1851 1867 1969 2013 2029 2115 2175 2274 2318 2111 2244 2285 2315 2431 2489 2519 2605 2693 2792 2850 1067 1134 1154 1169 1256 1271 Hex5HexNAc5DeoxyHex Hex6HexNAc5 NeuAcMeHex5HexNAc4DeoxyHex 1358 Hex6HexNAc5DeoxyHex NeuAcMe2Hex5HexNAc4 NeuAcMeHex5HexNAc5DeoxyHex Hex7HexNAc6 2394 2420 2623 2966 3212 NeuAc2MeHex5HexNAc4DeoxyHex NeuAc2MeHex5HexNAc5DeoxyHex NeuAcMe2Hex6HexNAc5 NeuAc2MeHex6HexNAc5DeoxyHex NeuAc3MeHex6HexNAc5 2639 3241 2785 2944 3416 3603 284 .
4 3 Moderate present.6-linked N-acetylglucosamine supports the presence of core α1. Since linkage data indicated an increase of 3. indicating that the Gal residues were in normal β-linkages.4-linked N-acetylglucosamine correspond probably to low amounts of α3-fucosylation.6-linked N-acetylglucosamine. 1983). the N-glycans were efficiently degalactosylated. the molecular ion at m/z 1079 (Fuc1Hex3 HexNAc2) disappeared. and 1705 (Hex4HexNAc5) and were more intense in the serum N-glycome of the patient suffering from cirrhosis. NeuAc. but differences in the relative intensities are found: (1) terminal mannose. ᭛.4. Y. and also contributes to the increase of terminal N-acetylglucosamine. m/z values refer to [M+Na]+ ions of methylesterified N-linked glycan. indicating the presence of neutral or incompletely sialylated complex-type oligosaccharides. Thus. Routine chemistry and clinical data of the patients X.. and terminal N-acetylglucosamine have more elevated relative intensities. 1543 (Hex3HexNAc5).51 13. and one molecular ion was observed at m/z 933 (Hex3HexNAc2) after 8-h digestion.8–5. 1745 (Hex3HexNAc6). respectively.6-linked mannose is present as traces and indicates the rarity of bisected complex structures. indicating that the GlcNAc residues were in normal β-linkages. Similar linkages are found in the serum N-glycome of patients with cirrhosis. continued m/z A B C Chemical composition Structure 3090 3777 NeuAc3MeHex6HexNAc5DeoxyHex 3309 3614 4052 4413 NeuAc3MeHex7HexNAc6 NeuAc4MeHex7HexNAc6 Symbols for the structural formulae are defined as follows: ■.92 1. ❍. two major molecular ions were observed at m/z 933 (Hex3HexNAc2) and 1079 (Fuc1Hex3HexNAc2). However. possibly due to steric hindrance.Total serum N-glycome in human diseases Table I. N-acetyl-D-glucosamine. A. These exoglycosidase digestions have been performed during 6 or 8 h. Exoglycosidase digestions To define the anomeric configurations as well as to confirm tentative sequences. (4) 4. (2) 3. and α-fucosidase treatment (Figure 3E and F).14 2. 1689 (Fuc1Hex3HexNAc5). Table II.1) Immunoglobulin G (g/dL) (n = 0.69–1. In addition. These data confirm an increased relative amount of oligosaccharides with an α6-fucosylated core in the serum Nglycome of the patient suffering from cirrhosis. 1851 (Fuc1Hex4HexNAc5). Therefore. ᭝.94 1. The MALDI-MS spectra (Figure 3A and B) present two major differences. ٗ.6-fucosylation. m/z values refer to [M+Na]+ ions of permethylated N-linked glycan. and traces of 3. mannose. C.6-linked mannose is present in considerable amount and suggests the existence of bisected complex-type oligosaccharides. After β-galactosidase and β-N-acetylhexosaminidase treatment (Figure 3C and D). ions at m/z 1689 (Fuc1Hex3HexNAc5) and 1851 (Fuc1Hex4HexNAc5) are more intense in the serum N-glycome of the patient suffering from cirrhosis (data not shown). galactose. After β-galactosidase. After β-galactosidase treatment.4. indicating the presence of an increased relative amount of oligosaccharides with an α6-fucosylated core. and 1892 (Fuc1Hex3HexNAc6). terminal galactose. the bisecting N-acetylglucosamine is partially resistant to the β-N-acetylhexosaminidase (Yamashita et al. m/z values refer to [M+2Na]2+ ions of permethylated N-linked glycan.3 3 Moderate Z 22 16 3.6-linked Man in the serum N-glycome of the patient suffering from hepatic fibrosis. after 6-h digestion. at m/z 1340 (Hex3HexNAc4) and 1486 (Fuc1Hex3HexNAc4).4. the MALDI-MS spectra of the N-glycans from a normal human serum (Figure 3A) and from a patient suffering from cirrhosis (Figure 3B) were characterized by the presence of seven molecular ions at m/z 1340 (Hex3HexNAc4). (3) the intensity of terminal fucose is increased in the same proportion as 4. and Z X Aspartate aminotransferase (U/L) (n =10–40) Alanine aminotransferase (U/L) (n = 10–40) Albumin (g/dL) (n = 3. fucose.91 51. after 6-h digestions. these exoglycosidase data confirm an increase of bisected complex-type N-glycans.4) Total bilirubin (mg/dL) (n < 1) Fibrosis Inflammation 64 36 2. 285 .56 0. Only the spectra obtained after 8-h digestion are shown in Figure 3. and (5) 3. β-N-acetylhexosaminidase. 1543 (Hex3Hex NAc5). The major molecular ion in the MALDI-MS spectrum of the Nglycans of the healthy subject and of the subject suffering from hepatic fibrosis was observed. desialylated N-glycans released by PNGase F from normal human serum and from patients suffering from cirrhosis were subjected to digestion with β-galactosidase alone or in combination with β-N-acetylhexosaminidase and α-fucosidase directly on the plate according to Mechref and Novotny (1998).4 4 Severe Y 73 30 2. other molecular ions were still observed at m/z 1340 (Hex3HexNAc4). 1486 (Fuc1Hex3HexNAc4). B.
4-Linked mannose 2. fragmentation was as described by Weiskopf et al. 145. acetylated. 161. 129.77 0. 162. 278 129.W. 102.05 0. The first stage (MS1) yields resolution of the methylated glycan mixture (Figure 4A) and gives the sugar chemical composition of each isobaric oligosaccharide (Table 1). The MS1 was characterized by the presence of abundant [M+2Na]2+ ions at m/z 1047 (data not shown). 190. 162.04 0. Desialylated and methylated normal serum N-glycome was analyzed. Fig. 118. 233 117. When analyzed in this system. The second and third stages (MS2 and MS3) give information on the nature of the antennae. with the Y4α or Y4β ions resulting from the loss of disaccharide antenna at m/z 1607.00 0. the permethylated N-glycans were analyzed using ESI-IT-MS.50 0. 175 102.19 0.38 1. 129. 662. Table III.01 0. and analyzed by gas chromatography MS.25 29.5 for the singly sodiated.00 0.24 21.14 0. 205 89. and at m/z 1143. The nomenclature describing the fragmentation of carbohydrates is that introduced by Domon and Costello (1988).45 0.14 38. 190 118. MS2 of ions m/z 1047 and MS3 of ions at m/z 1143. 346 117.04 1.6-Linked mannose Terminal GlcNAc 4-Linked GlcNAc 4-Linked HexNAc 3. 189.6-Linked mannose 3.08 0.50 37. 261 Assignment Terminal fucose Terminal mannose Terminal galactose 2-Linked mannose 4-Linked glucose 3-Linked galactose 6-Linked galactose 2.19 0. 130.4.07 B 0. the core structure. All glycans were detected as their [M+2Na]2+ except oligosaccharide (Hex3HexNAc2 DeoxyHex1) which was detected by the ions at m/z 929 ([M+2Na]2+) and 1836 ([M+Na]+). The analytical capabilities of ESI-IT-MS allow to analyze oligosaccharides as complex mixture and also offer multiple stages of fragmentation (MSn) to elucidate their structural heterogeneity.2.3 corresponding .23 The 80% acetonitrile fractions from Sep-Pak purifications of permethylated glycans were hydrolyzed. the branching pattern.6-Linked GlcNAc A 0.18a 24.22 0.42 32. 203. The MS3 spectrum of Y(4α. normal total serum N-glycome. 866.08 0.5 for the loss of both antennae. 161.34 0. 2.36 20. 130. N-glycome of patient with decompensated cirrhosis.13 0. at m/z 815. 205. GC-MS analysis of partially methylated alditol acetates obtained from the PNGase F released N-glycans Relative abundance Retention time (min) 15. 131. a Signals more intense after chemical desialylation of N-glycans.2.27 31.02 25.4β).2 and 639. and the 286 core fucosylation.59 30. B. 233 117.42 0.01 0. 118. 233 129.4-Linked GlcNAc 4.5 for the doubly sodiated.14 0. Morelle et al. 162. 333 117.24 Characteristic fragment ions 115. 189. 159.05 0. 129.04 0. A. b Signals not observed after chemical desialylation of N-glycans. 159. 189. 102.4β)2 ions gave all the characteristic fragment ions at m/z 939.42 35. 233 130.25b 28. 118. 190 118.32 25. MALDI-TOF MS analysis of permethylated total serum N-glycome from a cirrhotic patient.05 0. 205 117. 161. Other observed ions corresponded to [B5]2+ (B5/Y4α. 162. 159. 234 188. reduced.06 0. permethylated N-glycans form sodiated molecular adducts. 159.5 were characteristic of a biantennary complex Nglycan.09 0.12 0. 159. 129.09 35.06 0. 233 118. ESI-IT-MS of human serum N-glycome After desialylation.6-Linked mannose 3. (1998). 118.23 0. 234 99. Indeed.09 0.24b 27.
the more heterogeneous population of N-glycans observed in the MALDI-TOF MS analysis was confirmed (Figure 4A) and characterized by a relative increase of fucosylated oligosaccharides. The MS3 spectrum of ions at m/z 1318. MS3 of Y(4α.7.1 corresponding to Y3 and B5 fragment ions.8. The spectrum corresponds to a biantennary. D. 1998). these results show that the N-glycan is an agalactobiantennary complex-type oligosaccharide (Figure 4B and C).3 corresponding to the loss of an N-acetylhexosamine (Figure 4B). and Y4α. core-fucosylated N-glycan.4β.3β. 1592. In the MS spectrum.5. and [B5]2+ were observed at m/z 486. 900.4γ. β-N-acetylhexosaminidase.3 is typical of a fucosylated pentasaccharide core (Weiskopf et al. B5. and 1132.4. The MS2 spectrum of ions at m/z 1271. and C4 ions (data not shown). Y(4α.4β)2.6 corresponding respectively to B5/Y(4α. E) and from cirrhotic patient Y (B.9.5 corresponding to the chemical composition of Hex5HexNAc4 DeoxyHex1 was characterized by four major fragment ions at m/z 865. respectively to Y3α. corresponding to the chemical composition of Hex6HexNAc5. F) (A. The ESI-IT-MS analysis of the desialylated permethylated serum N-glycome of the patient with decompensated cirrhosis is shown in Figure 4. C.9 correspond to the second Nacetylhexosamine removal. The presence of the B4 ions at m/z 1385. and 1781.6 and 852. E.1 (Hex3HexNAc4DeoxyHex1) or of the doubly charged at m/z 929 is characterized by intense ions at m/z 1577. B5/Y4α.Total serum N-glycome in human diseases Fig. B5/Y3α. The ions at m/z 1318.7.4β. The spectrum corresponds to the fragmentation pattern of a triantennary complex N-glycan.0 corresponding to the core resulted in the presence of only two ions at m/z 939.. 3.4β ions.4γ)2]2+. corresponding to the chemical composition of 287 .3 indicates the core fucosylation. 902. B: β-galactosidase treatment.4β]2+. and 807.4β.4γ]2+. F: β-galactosidase.4γ)2. D: β-galactosidase and β-N-acetylhexosaminidase combined treatment. The MS2 spectrum of [M+Na]+ ions at m/z 1836. (1998) (Figure 4D and E). The MS2 spectrum of the [M+2Na]2+ ions at m/z 1256.5.4β)2.5.4β. The MS2 spectrum (Figure 4F) of the [M+2Na]2+ ions at m/z 1154. 1317. The MS2 spectrum of [M+2Na]2+ ions at m/z 1134. Taken together.8 corresponding respectively to [Y4α.3β. MALDI-TOF MS analysis of exoglycosidase digestions of total human serum desialylated N-glycome from normal subject (A.4β.7 and the MS3 spectrum of the Y3/Y(4α.4γ)3 ions at m/z 1129. [Y4α.1. C.2. and [Y(4α.3 are typical of a core-fucosylated biantennary complex N-glycan with a bisecting GlcNAc residue as described by Weiskopf et al. was characterized by the fragment ions at m/z 1039. Other characteristic ions B2α.4β)2 ions at m/z 1303. MS2 was performed on all major ions. and α-fucosidase combined treatment). Y(4α.2β.
7. representing the first three residues at the oligosaccharide reducing end. (E) MS3 of isolated ions at m/z 1303.1. we condensed the abbreviation as Y4α. (G) MS2 of doubly charged parent ions at m/z 1169. (C) MS3 of the isolated pentasaccharide core of parent ions at m/z 1836.7. (A) MS1 of the permethylated oligosaccharides. ESI-IT-MS analysis of total human serum N-glycome of a cirrhotic patient.3 from the doubly charged parent ions at m/z 1257. Morelle et al.7. (F) MS2 of doubly charged parent ions at m/z 1154. Fig. 288 .0. (B) MS2 of singly charged parent ions at m/z 1836.β. 4.W. (D) MS2 of doubly charged parent ions at m/z 1256. with a modification: instead of describing a fragmentation as Y4α or Y4β. The nomenclature describing the fragmentation of the carbohydrate is that introduced by Domon and Costello (1988). (H) MS2 of doubly charged parent ions at m/z 1359.1.1.
5. the periphery defined by α1.. Characterization of N-linked glycans of serum N-glycome isolated from patients with decompensated cirrhosis The distribution of the N-glycans is more heterogeneous and is characterized by the following: (1) the existence of an important subpopulation of neutral and truncated biantennary complex oligosaccharides (ions at m/z 1486. (2004). and (2) the presence of a bisecting GlcNAc residue in the intense ions at m/z 2318 and 2630. 1999. ESI-IT-MS allows the structural characterization of N-glycans present in the mixture. for singly and doubly charged ions.. and 1867. 2003). Butler et al. 2003. still it provides the opportunity of using these 26 groups of N-glycans as prognosis or diagnosis markers.6-Linked N-acetylneuraminic acid predominates. and any informative MS3 was obtained. α2. Flahaut et al. Serum N-glycome was partly characterized by Callewaert et al. Mills et al.2 and 923. was characterized by the loss of antennae observed with Y4α fragment ions at m/z 1822.3-fucosylated compounds seen in the linkage analysis and present in smaller amounts. They partly reflect the extreme heterogeneity of total serum N-glycome.. In this study. and it is adapted to clinical laboratory. In this study.. It has been 289 . 1689.and α1. Unfortunately. The number and position of the antennae.and α2. 1851. based on the increase of 3.. as demonstrated by ESI-IT-MS. Callewaert et al. 1543. the MALDI-TOF approach only resolves this heterogeneity as a mixture of isobaric compounds. All three different phases of the protocol–PNGase F digestion.1. but there is an uncertainty concerning the exact position of the N-acetylhexosamine residue. 26 N–glycans have been characterized. As previously reported. purification of released oligosaccharides via solid-phase extraction on graphitized carbon column. 1689. Y4α/Y3γ. suggesting a triantennary fucosylated structure. 2004).. 1648. we have defined the major ones.and 2. 1705. 2002.3 is approximately 5 (Table 3). 2003. the ratio of α2.. bisecting GlcNAc. and (3) the relative increased core fucosylation. The MALDI-TOF approach allows partial identification of the sialylated and neutral glycans through their sugar composition.6linked mannoses corresponding to triantennary oligosaccharides and on ESI-IT-MS results. the same modification is probably present.0 (Figure 4H) indicates the presence of the three N-acetyllactosamine units and the core substitution by a fucose residue. The MS2 spectrum of the [M+2Na]2+ ions at m/z 1169. indicating either a biantennary structure with terminal Nacetylhexosamine residue.7 (Figure 4G) indicates the presence of two Nacetyllactosamine units. and a bisecting GlcNAc. Total serum N-glycome in patients with cirrhosis is characterized by the presence of an important population of Nglycans with bisected GlcNAc (Callewaert et al. These authors have defined 11 desialylated oligosaccharides depending on their electrophoretic mobility and exoglycosidase digestions. 1999) and can be expressed as relative intensity. as deduced from the GC-MS analysis. 2003. 2004. the MS3 spectrum of the ions at m/z 1304 was not enough informative to decide between these two isobaric structures.. with the restriction of isobaric compounds and the sensitivity of the technique that did not enable us to identify α1.7 ions. Traces of α1. It is therefore fully suitable for the search of Nglycosylation modifications in congenital or acquired diseases and also for the comparison between the different states of a disease.6 to α2. Ito et al. glycans possessing a bisecting GlcNAc residue are present as very minor components.3-linked fucose observed in the GCMS analysis (Table 3) were not detected by ESI-IT-MS.6 linked.6-linked mannose associated with the intensities of 2. the N-glycan mix constituting total serum N-glycome was elucidated by MS analyses of desialylated or methylesterified oligosaccharides and finally using permethylation. and this analysis can be achieved on less than one microliter of serum. and 1851). The structure of all N-glycans is summarized in Table 1. a N-acetyllactosamine. Modifications in the expression of N-acetylglucosaminyl transferases III and V (GnTIII and GnTV) are associated with many physiological and pathological processes in the liver (Taniguchi et al. should result in hundreds of different N-glycan structures. Characterization of N-linked glycans of normal human serum N-glycome Complex-type biantennary oligosaccharide represents the major component of healthy subject N-glycome. and is very close to the ratio observed in transferrin (Fu and van Halbeek. 2001).4. we worked on less than 2% of the initial sample.4. Qualitative and semi-quantitative aspects of the N-glycome observed by MALDI-TOF are in agreement with previously observed results on various glycoproteins isolated from serums (Wilson et al.9 and 793. Y4β fragment ions at m/z 1563.Total serum N-glycome in human diseases Hex5HexNAc4DeoxyHex1. Among the fucosylated oligosaccharides. 1992). 1810.3. Trace amounts of neutral oligosaccharides are observed corresponding to agalactosylated biantennary complex glycans. Finally. Discussion We have developed a rapid protocol that can provide total serum N-glycome in less than 24 h. or a triantennary complex oligosaccharide with two agalactosylated antennae. as deduced from the relative intensities of ions at m/z 1664 and 2029 (Figure 1B).6-linked N-acetylneuraminic acid.6-linked fucose residues and by α2. a complementary structural analysis involving permethylation makes it possible to define the main changes.3. The same ambiguity was also found in the MS2 of 1154. most of the fucose residues are core α1. We are currently assessing this protocol to define prognosis and diagnosis markers in hepatic cirrhosis and hepatocarcinoma. 1502. and either methylesterification or chemical desialylation–enabled the development of a high-throughput method with full automation. respectively. Also. the B5 ions indicate the core fucosylation. Interpretation of the MS2 spectrum of [M+2Na]2+ ions at m/z 1359. the ratio of biantennary to triantennary is about 6:1. In ions at m/z 1543. The signal strength of each ionized glycan reflects the relative amount of material present in the sample (Harvey.
the aspartate aminotransferase (AST)..000 U/mL) for 3 h. relates to the logarithmic ratio of the relative intensity of two N-linked oligosaccharides. Fujii et al. Total N-glycome is the sum of all serum glycoprotein glycosylations.1%) (Packer et al. Higai et al.. From the sialylated or unsialylated N-glycome. 1992) resulting in the hyperasialoglycoproteinemia observed in cirrhosis and hepatocellular carcinomas (Sawamura et al. for 2 h (Varki and Diaz. and transferrin (Block et al... 2003). Release and purification of the serum N-glycome Serums (10 μL) were denatured at 100°C in the presence of sodium dodecyl sulfate and β-mercaptoethanol. The presence of a relative increase of multiantennary difucosylated oligosaccharides in patient Y as compared with patients X and Z might correspond to the modifications of the degree of branching of α1-acid glycoprotein oligosaccharides observed during acute and chronic inflammation (van Dijk et al. New England Biolabs. v/v) containing trifluoroacetic acid (0. One possible hypothesis is that the neutral oligosaccharides are present on pro-inflammatory galactose-deficient immunoglobulins. Serum albumin was measured with the bromcresol green method. which are found in numerous diseases (Moore et al. curiously. Affinity chromatography purification of cirrhotic patient immunoglobulins will confirm the origin of the neutral oligosaccharides observed. Patient Y was suffering from liver fibrosis with acute alcoholic hepatitis. In particular. and only quantitative modifications were observed in cirrhotic patients by highresolution two-dimensional electrophoresis (Gravel et al. shown that fatty liver is associated with an increased risk of progression to cirrhosis. Moore et al. and the remaining part was chemically desialylated.. the protein N-glycanase F (PNGase F.. α1-antitrypsin. and Patient Z was suffering from chronic hepatitis C. these N-glycans either result from altered biosynthesis or from the dysfunction of the asialoglycoprotein receptor (Burgess et al. obtained after desialylation of the N-glycome.. . To define what proteins are modified. IgG were measured by nephelemetry on a BN2 (dade Behring. 1984). Templemars. 2005). Esterification of sialic acids To stabilize the sialic acid moiety under MALDI-MS conditions. Alterations in the expression of GnTIII and FUT8 are probably one of the primary events leading to the lipid accumulation in hepatic steatosis. 2 M at 80°C. the isoelectrophoretic behavior should be modified since the loss of terminal sialic acids leads to a cathodic shift of the isoelectric point of glycoproteins. The point of developing a sensitive assay for screening modification of total serum N-glycome is to obtain diagnosis 290 and prognosis markers for chronic diseases such as the GlycoCirrhoTest (Callewaert et al. will precise the N-glycome modifications observed during inflammation.6-fucosylated oligosaccharides..6-fucosyltransferase (FUT8) has also been involved in steatosis in the liver because of a downregulation of lysosomal acid lipase by the remodeling of its glycosylation (Wang et al.W. a positive correlation was observed between the relative intensity of ions at m/z 1486 and the immunoglobulin G (IgG) concentration (data not shown).. 1995.. Control serums were obtained from healthy volunteers. 1996). and the grading and staging was done according to the METAVIR scoring system (The METAVIR cooperative group. This test. Fibrosis and inflammation were evaluated after liver biopsy. 1996). corresponding in our study to ions at m/z 2013 and 2029. and the neutral and the sialylated oligosaccharides were eluted together with an acetonitrilewater solution (25/75. France). the isoelectrophoretic pattern of transferrin should be close to the one observed in CDG Type II. GnTIII is involved in the dysfunction of the apolipoprotein metabolism. Morelle et al. Proteomic approaches have been used in liver cancer. the total bilirubin. complementary studies need to be done. resulting in lipid accumulation that leads to fatty liver. Finally. 1984). one of the first steps of alcohol-induced liver injury (Ihara et al. 1998. Patient X was suffering from decompensated cirrhosis with acute alcoholic hepatitis. where the increase in fucosylation has been associated with αfetoprotein. of additional patients with various inflammatory diseases treated or not. 2001). and the tests were performed on an Olympus AU600. 1985. UK) was added (1 μL) (500. After the addition of nonidet-P 40. we currently investigate into all the mathematical combinations offered by this MALDI-TOF MS approach.. Lee et al. 1998). The oligosaccharides were purified by solid-phase extraction on a graphitized carbon adsorbent (Alltech Associates.. to define new noninvasive biomarkers. The oligosaccharide structure corresponding to ions at m/z 1486 (Table 1) is present on immunoglobulin (Parekh et al. The second characteristic of the Nglycome is the relative increase in α1.. 2004). 2004). 2005). and the alanine aminotransferase (ALT) were measured according to the International Federation of Clinical Chemistry (IFCC) recommendations. Further investigations... If the neutral agalactosylated oligosaccharides were present on liver glycoproteins such as transferrin. α1-acid glycoprotein. Chemical desialylation Sialic acids were cleaved by treatment of N-glycan samples with acetic acid. 1994). Another major glycosylation change appearing in the N-glycome of patients suffering from hepatic cirrhosis is the presence of neutral agalactosylated oligosaccharides. Paris. 2005). the sialic acid residues of PNGase F-released oligosaccharides were stabilized by methylesterification of their carboxylic group (Powell and Harvey. Materials and methods Patient samples Serums were obtained from patient admitted in the Department of Gastroenterology and Hepatology of Lille University Hospital for decompensation of cirrhosis or hepatitis. these are normal for the three patients (data not shown). 1990. France). Herts. α1. Half of total N-glycan fraction was methylesterified. Therefore.
2 U in 100 μL of 100 mM ammonium acetate buffer. Romano. I. 300–306. Pennec. Biomed. The total ionic current (TIC) signal was acquired for 1 min. The isolation width was set to 2 U. 155.R.... San Jose. A.23. No sheath gas was used. Schollen.. After external calibration. Proc. English. Critchley. Y. D. EC 3.. U. the ‘dried droplet’ preparation technique was employed. This research was supported by the Centre National de la Recherche (Unité Mixte de Recherche CNRS/USTL 8576. St-Quentin Fallavier. MA) equipped with a pulsed nitrogen laser (337 nm) and a gridless delayed extraction ion source. J. London. Baenziger. Chrompak.52.1. Lowman. and Brown. Hayes. Hebestreit. Argenteuil. Clement. pH 4. Butler. Res.. G. A. EC 3. whereas one part out of fifty was used for methylesterified N-glycans. M. Sci. B. V..2. a pulse delay time of 200 ns. Pollacchi. electrospray ionization ion trap mass spectrometry. Albersheim... B.. Tennant. E. (1994). Linkage analysis of N-glycans Partially methylated alditol acetates were prepared from permethylated samples for GC-MS linkage analysis as described previously (Albersheim et al. the CNRS. (2000) Increased N-linked glycosylation leading to oversialylation of monomeric immunoglobulin A1 from patients with Sjogren’s syndrome. M.1. P.6.J. K. 10 mU in 100 μL of 50 mM ammonium formate buffer. Electrospray mass spectrometry Structural characterization of methylated N-glycan samples was carried out using electrospray ionization with an LCQ Deca XP+ ion trap mass spectrometer equipped with a nanoelectrospray ionization source (Thermo-electron. The Mass Spectrometry facility used in this study was funded by the European Community (FEDER). Basset. Scand. For desialylated oligosaccharides. 702–706. L. GC-MS.B. protein N-glycosidase F.Total serum N-glycome in human diseases Exoglycosidase digestions These were carried out on the released desialylated Nglycans using the following enzymes and conditions: Nacetyl-β-D-hexosaminidase (from jack bean.1. Samples were analyzed in delayed extraction mode using an accelerating voltage of 20 kV.. France).. Dueymes. France). H. Electron ionization spectra were recorded using ionization energy of 70 eV. MALDI-TOF MS. collision energies were set to 20–40% of maximum. For all measurements. France) according to Dell et al. E. 347–348. A. PNGase F. Nevins.. France). P. Director: Dr Jean-Claude Michalski) and the Ministère de la Recherche et de l’Enseignement Supérieur. Gas chromatograph was equipped with a CP-Sil 5CB/MS capillary column (25 mm × 0.. M.. T. R.. and Karr. Carchon.. 102. and the Université des Sciences et Technologies de Lille. Eur.5. Quelhas. Carbohydr. congenital disorders of glycosylation. Acknowledgments We are grateful to Prof. β-galactosidase (from bovine testes. between 100 and 200 scans were averaged for each spectrum shown. Detector bias gating was used to reduce the ion current below masses of ∼500 Da. For the generation of the MSn spectra. Roche Molecular Biochemicals.U..S. G. E.. gas chromatography mass spectrometry. Youinou. (2005) Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans. D. W.. Block. P. After derivatization.. Philippe Delannoy for comments on the manuscript. P. Fimmel. Newsome. Odense. Hepatology.M. P.. Jamin. IgG. Hibbert. pH 4. Vilarinho. 5. All experiments were performed in the positive-ion mode. C. (1992) Abnormal surface distribution of the human asialoglycoprotein receptor in cirrhosis.D. 340–345. 51. Therapondos. S. (2002) A preliminary evaluation of the differences in the glycosylation of alpha-1-acid glycoprotein between individual liver diseases. Matthijs. M. We thank Adeline Page for assistance with the mass spectrometer. the reaction products were purified on C18Sep-Pak (Waters Ltd. Permethylation of N-glycans Permethylation using the sodium hydroxide procedure was performed according to Ciucanu and Kerek (1984)..2.2. C. Blumberg... α-L-fucosidase (from bovine kidney. and Roitt. Chromatogr..1% of trifluoroacetic acid). Natl.. A.. 15.A. References Adamowicz. Immunol. A.6.. one part out of 100 was loaded on the target.F. pH 4. 16. Steel. The partially methylated alditol acetates were dissolved in methanol before on-column injection at 130°C. W.. (1967) A method for the analysis of sugars in plant cell wall polysaccharides by gas– liquid chromatography. Samples dissolved in a solution of 70% methanol and 1% formic acid were sprayed from a goldcoated medium-length borosilicate capillaries (Proxeon. St-Quentin en Yvelines. J. and 291 . Meylan. Abbreviations CDG.. H.5-dihydroxybenzoic acid (DHB) as matrix (10 mg/mL of DHB in an acetonitrile/ water solution [70 : 30] containing 0. EC 3. MALDI-TOF mass spectrometry All mass spectra were acquired on a Voyager Elite (DESTR) reflectron time-of-flight (TOF) mass spectrometer (Perseptive Biosystems. J. L.51.2 U in 100 μL of 50 mM ammonium formate buffer.R.32 mm. ESI-IT-MS.. and Smith. Framingham..T. Comunale.G..J..M... 0. Native and methylesterified Nglycans were co-crystallized with 2. The spray voltage was 1 kV and the capillary temperature was 170°C. GC-MS analysis were recorded using an Automass II 30 quadrupole mass spectrometer interfaced with a Carlo Erba 8000 Top gas chromatograph (Finnigan. 779–784. Evans. Teles. M. 1967). 365–372. J. Pediatr.. J. A. P. 0. The gas vector was at a flow rate of 2 mL/min... N. France). (1996) Carbohydrate deficient glycoprotein syndrome-like transferrin isoelectric focusing pattern in untreated fructosaemia. immunoglobulin G. Burgess. les Ullis. The GC oven was held at 130°C for 1 min before increasing to 180°C at 2°C/min and then to 240°C at 4°C/min. and a grid voltage of 66%..C.. Denmark). and Pronicka. Boyter. and others.F. C. Sigma). Anderson.. Durand.. CA). the Région Nord-Pas de Calais (France). Acad..A. matrix-assisted laser desorption ionization time of flight mass spectrometry. Sigma.
Y. Rapid Commun. (2003) Altered glycosylation of alpha1-acid glycoprotein in patients with inflammation and diabetes mellitus. R.. Jaeken. A. K. Grünewald. E.6 N-acetylglucosaminyltransferase is an early event in hepatocarcinogenesis. Ikeda. FSH and GH levels. 1H nuclear magnetic resonance spectroscopy.W... R.W... Casaer. Geysens.. Flahaut. AIDS. Glycoconj. 91. Hart. 52. H. 8. and Matsumoto. Anbergen. Vanhecke. and Eeckels. Nakada.. J. A. T.. E. Charlwood. Aoki.. Van der Stelt. Glycobiology. 911–915. Jaeken. 455–463. fast atom bombardment-mass spectrometry. M.H. Hachulla. Glycobiology. Kim. 227–233. Y.H. A. Panico. Callewaert. P. J. B.K. and Mestecky. Y. 78–85. Yamamura. and others. Takemura.. 13. Mills. Powell.C. Van Vlierberghe. 236–247. Sakon... Acad.. (2003) Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I. A. Sultan.. E.. Biophys.H. 657–666. J. 137. 631–637.. M. K.. Miura.. 13. Stanworth. B. 95. Nat. J... L.. A. Matthijs. and Kollberg. Res.B. (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Acta.... Mizuochi. Biochem. 351–357. P. and Tashiro. McDowell. Vanderschueren-Lodeweyckx.P. 34. J.. J. A. M. Callewaert. Moldoveanu. van Dijk.. and. Sheng. A. H.. N... Mills. S. Delanghe. T. and Harvey. B. Anal. Z... S. M.. Taniguchi. Hepatology. 179.. Lawson. C. Papac. Arch.. A. Glycoconj. 87. Clin. N. C.. (2001) Ectopic expression of alpha1. and Turner. Acta. Noda. (1984) Hyperasialoglycoproteinemia in patients with chronic liver diseases and/or liver cell carcinoma..T. Larsson. E. Nishiura.. 18. (1992) N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography. (2003) The effects of ethanol on the glycosylation of human transferrin.W.. N. R. Med. Sci... Ko.. J.. 275–281. Wakasa. (1993) Inflammation-induced expression of sialyl Lewis X-containing glycan structures on alpha. pp.. Cold Spring Harbor. 1-antitrypsin in congenital disorders of glycosylation type I is not random. 6009–6018. Ryden.. M. (1984) The release and the purification of sialic acids from glycoconjuguates: methods to minimize the loss and migration of O-acetyl groups. P. W. 117–125. Toyosawa. 381–389. R. Miyoshi. Anal. Ikeda...A. M.. G. Keir... R. (1998) A general approach to desalting oligosaccharides released from glycoproteins. New York. (1999) Acquired glycosylation changes in human disease. 1027–1032. 1-antichymotrypsin microheterogeneity in crossed immunoaffinoelectrophoresis with free concanavalin A: a useful diagnostic tool in inflammatory syndrome. Danel. 349–450. and Taniguchi. H. P. 265.. Harvey.... Shiozaki. Biochem. K. M. and Kerek.. J. Briggs. W. Glycoconj. (1996) New alterations of serum glycoproteins in alcoholic and cirrhotic patients revealed by high resolution twodimensional gel electrophoresis. Clin. 397–409.. G. 618–624. 73–85. Methods Enzymol. Cummings. (2003) The underglycosylation of plasma alpha. Yamamoto. and Marth. Chim. 70. Pediatr. A. D. Natl.S. F. M. Corbeel. and Jaeken. J. and Ihara. T. Y.E.. A. N.. 15.H.. 565–580.. Biophys. Ekuni. Wu. T. (1998) Increased serum concentrations of carbohydrate-deficient transferrin (CDT) in patients with cystic fibrosis. M. The French METAVIR. Chung...B. and Costello. L.. (eds). D. (2001) Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. 18–31.. E...A... Van Hecke. and others. B. and Jones.. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases. Goodarzi. Y. and Guimon. Int. M. 231–236. 452–457. Aubry. 13. 230. Yeom. partial TBG-deficiency.....W. 367–375. Higai. Kang.. T. and Contreras.. Schollen. 601–622. J. Honda. Reason. Tomana. Sutton. Johnson. Molemans. De Graaf. Biophys. and Winchester. 429–434.J.. 10. (1994) Mass spectrometry of carbohydrates-containing biopolymers. Anal. Hochstrasser. and van Dijk. Ito. (2003) Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis. (1998) Defective galactosylation of serum transferrin in galactosemia...... D. J. Miyoshi. Callewaert. Fu. 131.. 287–300. J. and Novotny. Chem. Packer. 103. Kulhavy.. 14. N. Pahlsson. Snoek. Res.. Havenaar. Brown.T. (1995) Alpha1-acid glycoprotein (orosomucoid): pathophysiological changes in glycosylation in relation to its function.. Y. (2002) Diagnostic accuracy of alpha(1)-acid glycoprotein fucosylation for liver cirrhosis in patients undergoing hepatic biopsy.. T. Balant. (1998) Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease. Walzer. P. Matsuta. 20. Mass Spectrom... (2005) Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals. Goepfert..Y. J. Carbohydr.H. 206. Ihara. Glycobiology.. Song. K. D. 10. F. Mechref. J. 2195–2201.R. I.. R. In Varki. Proc. Sameshima. C.. A. N. and Lindgren. N. Clayton. 426. 1-acid glycoprotein (orosomucoid) in human sera. K. Mass Spectrom. M. Fernandes. S.. Glycobiology.A. 2526–2530. N. W. R. Y. R. 15–20. Cancer. Ups. Hazama. and Brinkman-van der Linden. Taniguchi. and Diaz. 108–132.. Ijuhin..K... J. (1996) Stabilization of sialic acids in Nlinked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry.... A. 191–198. (1984) A simple and rapid method for the permethylation of carbohydrates. A.. 8. Monden. (1980) Familial psychomotor retardation with markedly fluctuating serum proteins. G. Takeda. Glycobiology. 12. A. J. Khoo. M. (1988) Alpha.T. Laroy.. A. Chung.. Cooperative Study Group (1994) Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Esko. Humbert. C. J.J. Li. Ko. E. Biochem. J. Chem. J. S. and Contreras. Jardine.. (2002) Congenital disorders of glycosylation: a review. E.6 fucosyltransferase in mice causes 292 ..L.. Glycobiology.. and Klein. U.S.. Varki.. Mian.. Res. Wang.H. Biochim. J. Biol.E.. A. Res.. Y. (1988) Systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjuguates. and Contreras. W. Asialoglycoprotein receptor in cirrhosis and liver cell carcinoma. and Taniguchi... Y... Parekh. B. A. F. (1998) Ectopic expression of N-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid storage. A. 5. 737–747.V. and Hayem. H.. Domon.. Laine. M.. 177. Yersin. Ohnishi. A. Flodin.H. Commun. Morelle et al... Lee. 1217–1221. Glycoconj. D. A. and others. Moore. Freeze.. Y.. 329. G. 13. Gastroenterology.. Clayton.. X. T. T.. T. Dell...W. H. K.. Rademacher. 15. and van Halbeek. S. (1990) Structural heterogeneity of sugar chains in immunoglobulin G. Gravel. Miyoshi. A.. Eggermont.. J.. 19.A. S. Tsujimoto. J. R. M. 48.. (2004) Hyperexpression of N-acetylglucosaminyltransferase-III in liver tissues of transgenic mice causes fatty body and obesity through severe accumulation of Apo A-I and Apo B.J. S. Y. Y. E.. S. 220. Nishikawa. Fujii.. D. N.. Nishikawa.. increased serum arylsulfatase A and increase CSF protein: a new syndrome? Pediatr. 316. Yoshimura. D.. 1455. Essentials of Glycobiology.F. Exp. Chem. Novak. K. Sawamura.B. Miyagawa.. M. Y. P. N. H..J.I.G.. J. (1999) Implication of N-acetylglucosaminyltransferases III and V in cancer: gene regulation and signaling mechanism. (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Taniguchi. R. and Morris. K.I.I. H. 469–475.. J.. Michalski. and Winchester. Med. S. Matthijs. Chin.. Dwek. Rev. Clin. and Kim.G.S.. Sci..C. J. Ito.. E. Chem. Leung. R... (2004) Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. 445–454. J. 11. Med. others. E. P. (1998) Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins. J. Mian.. W. Ciucanu. Varki.A. 53–63. E.. I. H.J..C. T. Nature. Miyoshi.. M. Y.. Suzuki..R.. L.. Glycobiology..W. K. (2001) Elevated expression of UDP-N-acetylglucosamine: alphamannoside beta1. 209–217. C. and Redmond. Biochem. Azuma. (1998) A highthroughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. D.
. A.L. N.. N.. A. Katunuma. N.S. Matsuda.L. 11. Chem.. Iwaki.... and Kobata. P. and Kobata. A.H. An extremely heterogeneous pattern enriched with nonreducing terminal N-acetylglucosamine residues. Y. Koide. (1998) Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides. and Packer. (1989) Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. K. Karlsson. 70... Chem.Total serum N-glycome in human diseases steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase. Proteome Res. (2002) Sequential analysis of N..and O-linked glycosylation of 2D-PAGE separated glycoproteins.. Chem. B. 1098–1107. (1983) Structural studies of the carbohydrate moieties of rat kidney gamma-glutamyltranspeptidase. J. D. A. 293 . Glycobiology. 4441–4447 Wilson. 264. Y. Biol. 165–174.. N.. Yamashita. 1. 521–529.. J. N. J.J. T. A. 2415–2423.G. Weiskopf.. Biol. K. Vouros.. Yamashita. Anal. Endo. and Harvey. 258.. Hitoi. Tsujii. Schulz.
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