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1.
Xiong L Regnier FE 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,782(1-2):405-418
The purpose of the work described in this paper was to develop a new approach to the identification of glycoprotein with particular types of glycosylation. The paper demonstrates N-glycosylation sites in a glycoproteins can be identified by (1) proteolysis with trypsin, (2) lectin affinity selection, (3) enzymatic deglycosylation with peptide-N-glycosidase F (PNGase F) in buffer containing 95% H(2)(18)O, which generates deglycosylated peptide pairs separated by 2 or 4 amu, (4) reversed-phase separation of the peptide mixture and MALDI mass analysis, (5) MS-MS sequencing of the ion pairs, and (6) identification of the parent protein through a database search. This process has been tested on the selection of glycopeptides from lactoferrin and mammaglobin, and the identification of the ion pairs of fetuin glycopeptides. Glycosylation sites were identified through PNGase hydrolysis in H(2)(18)O. During the process of hydrolyzing the conjugate, Asn is converted to an aspartate residue with the incorporation of (18)O. However, PNGase F was observed to incorporate two (18)O into the beta-carboxyl groups of the Asp residue. This suggests that the hydrolysis is at least partially reversible. 相似文献
2.
Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed. 相似文献
3.
Joseph V. Lomino Andreas Naegeli Jared Orwenyo Mohammed N. Amin Markus Aebi Lai-Xi Wang 《Bioorganic & medicinal chemistry》2013,21(8):2262-2270
A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery. 相似文献
4.
Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei 总被引:1,自引:0,他引:1
Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei. 相似文献
5.
Hägglund P Matthiesen R Elortza F Højrup P Roepstorff P Jensen ON Bunkenborg J 《Journal of proteome research》2007,6(8):3021-3031
Global proteome analysis of protein glycosylation is a major challenge due to the inherent heterogeneous and diverse nature of this post-translational modification. It is therefore common to enzymatically remove glycans attached to protein or peptide chains prior to mass spectrometric analysis, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide-N-glycosidase (PNGase) digestion, with concomitant deamidation of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-beta-N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond between the two N-acetylglucosamine (GlcNAc) residues in the conserved N-glycan core structure, leaving single GlcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (beta-galactosidase, neuraminidase and N-acetyl-beta-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides were also identified with a single N-acetylhexosamine attached, arguably due to partial deglycosylation of O-glycan structures by the exoglycosidases used together with Endo D/H. 相似文献
6.
Fujihara J Hieda Y Xue Y Nakagami N Takayama K Kataoka K Takeshita H 《Biochemistry. Biokhimii?a》2006,71(Z1):S65-S70
Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid
(mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat
germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described.
This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary
gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation
either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated
on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo
H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding
with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while
that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I
exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of
the enzyme. 相似文献
7.
T Haraguchi S Fisher S Olofsson T Endo D Groth A Tarentino D R Borchelt D Teplow L Hood A Burlingame 《Archives of biochemistry and biophysics》1989,274(1):1-13
Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established. 相似文献
8.
从脑膜炎脓杆菌(Flavobacterium meningosepticum)基因组中通过PCR扩增了N-糖酰胺酶F(PNGase F)基因,经酶切后与表达载体pET28a连接,获得的重组质粒转入大肠杆菌BL21(DE3)。重组大肠杆菌经诱导表达和纯化提取后,获取大量高纯度N-糖酰胺酶F,其纯度达90%以上。试验证明,经纯化的重组N-糖酰胺酶F可以切除核糖核酸酶B、转铁蛋白和人IgG等糖蛋白上的N-糖链,具有脱糖基化作用。 相似文献
9.
The synthesis of alkyl-alpha-rhamnosides by alpha-rhamnosidase was studied using rhamnose and rhamnosides, particularly the flavonoid naringin, as glycosylation agents, and water soluble alcohols as acceptors. The reaction products were analyzed by HPLC chromatography and identified by 13C y 1H NMR. The glycosylation of alcohols by reverse hydrolysis was maximum for 40% methanol, 30% ethanol, 10% propanol and 20% isopropanol. Under optimum conditions the yield of rhamnose to alkyl-alpha-rhamnoside transformation decreased from 68% for methyl-alpha-rhamnoside to 10% for isopropyl-alpha-rhamnoside. The time course of rhamnosylations produced using naringin as the donor was comparable with that of the reverse hydrolysis obtained at the same molar concentration of the donor. The flavonoids and their derivatives remaining in the solution after the glycosylation were removed by ion exchange QEAE chromatography at pH 10. These results indicate that both, reverse hydrolysis and glycosylation by naringin are acceptable procedures for the enzymatic synthesis of short chain length alkyl-alpha-L-rhamnosides. 相似文献
10.
J. Fujihara Y. Hieda Y. Xue N. Nakagami K. Takayama K. Kataoka H. Takeshita 《Biochemistry. Biokhimii?a》2006,71(1):S65-S70
Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme. 相似文献
11.
Aedes aegypti chorion peroxidase (CPO) plays a crucial role in chorion hardening by catalyzing chorion protein cross-linking through dityrosine formation. The enzyme is extremely resistant to denaturing conditions, which seem intimately related to its post-translational modifications, including disulfide bond formation and glycosylation. In this report, we have provided data that describe a new type of glycosylation in CPO, where a mannose is linked to the N-1 atom of the indole ring of Trp residue. Through liquid chromatography/electrospray ionization/tandem mass spectrometry and de novo sequencing of CPO tryptic peptides, we determined that three of the seven available Trp residues in mature CPO are partially (40-50%) or completely mannosylated. This conclusion is based on the following properties of the electrospray ionization/tandem mass spectrometry spectra and the enzymatic reaction of these peptides: 1) the presence of a 162-Da substituent in each Trp residue; 2) the presence of abundant fragments of m/z 163 ([Hex + H]) and [M + H - 162] (typical for N-glycosides); 3) the absence of a loss of 120 Da (this loss is typical for aromatic C-glycosides); and 4) the cleavage of the glycosidic linkage by PNGase A or F (typical for N-glycans). These results establish that a C-N bond is formed between the anomeric carbon of a mannose residue and the N-1 atom of the indole ring of Trp. This is the first report that provides definitive evidence for N-mannosylation of Trp residues in a protein. In addition, our data demonstrate that PNGase can hydrolyze Trp N-linked mannose in peptides, which is unusual because no typical beta-amide bond is present in the Trp-mannosyl moiety. Results of this study should stimulate research toward a comprehensive understanding of physiology and biochemistry of Trp N-mannosylation in proteins and the overall biochemical mechanisms of PNGase-catalyzed reactions. 相似文献
12.
为了优化利用N糖苷酶F(PNGase F)酶解单克隆抗体中N糖的方法,应用本公司生产的单抗对PNGase F酶的酶解条件进行优化,包括缓冲液pH、酶种类、仪器、酶解程度、变性缓冲液及酶加入量等,总结酶解条件对N糖谱结果的影响。结果显示,置换缓冲液至1×PBS中可以避免某些单抗在低pH酶解时G0F转化为G0F-GN并可改善峰型;快速PNGase F和加入变性缓冲液能有效提高酶解效率;UPLC和1.7 μm粒径色谱柱能提高分离度;不完全酶解影响N糖含量结果。研究结果表明,采用优化的酶解条件可快速、有效的酶解单抗上的N糖,使N糖谱结果准确可靠,为细胞株筛选和单抗药物质量控制提供有效手段。 相似文献
13.
Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion-exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full-length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F. The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density-gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active-site domain of the enzyme. 相似文献
14.
F March C Enrich M Mercader F Sánchez C Mu?oz P Coll G Prats 《Experimental parasitology》1991,73(4):433-439
Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded. 相似文献
15.
Palmisano G Melo-Braga MN Engholm-Keller K Parker BL Larsen MR 《Journal of proteome research》2012,11(3):1949-1957
N-Linked glycoproteins are involved in several diseases and are important as potential diagnostic molecules for biomarker discovery. Therefore, it is important to provide sensitive and reliable analytical methods to identify not only the glycoproteins but also the sites of glycosylation. Recently, numerous strategies to identify N-linked glycosylation sites have been described. These strategies have been applied to cell lines and several tissues with the aim of identifying many hundreds (or thousands) of glycosylation events. With high-throughput strategies however, there is always the potential for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyzes the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site. We have evaluated this common large-scale N-linked glycoproteomic strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery found in mammalian systems and some pathogenic microbes. After isolation and proteolytic digestion of E. coli membrane proteins, we investigated the presence of deamidated asparagines. The results show the presence of deamidated asparagines especially with close proximity to a glycine residue or other small amino acid, as previously described for spontaneous in vivo deamidation. Moreover, we have identified deamidated peptides with incorporation of (18)O, showing the pitfalls of glycosylation site assignment based on deamidation of asparagine induced by PNGase F in (18)O-water in large-scale analyses. These data experimentally prove the need for more caution in assigning glycosylation sites and "new" N-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Besides showing the spontaneous deamidation, we provide alternative methods for validation that should be used in such experiments. 相似文献
16.
Treatment of [3H]glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-Mr (49K) product. For [3H]mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an additional size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90env, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80env, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. Similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicating that O-linked glycosylation is a conserved feature of retroviral env proteins. 相似文献
17.
Wagner-Rousset E Bednarczyk A Bussat MC Colas O Corvaïa N Schaeffer C Van Dorsselaer A Beck A 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,872(1-2):23-37
Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety. 相似文献
18.
X. S. Zhang G. B. Proctor J. R. Garrett D. K. Shori G. H. Carpenter 《Glycoconjugate journal》1996,13(1):91-98
The glycosylations of five different rat submandibular kallikreins, rK1, rK2, rK7, rK9 and rK10, vacuum-blotted onto nitrocellulose membranes, have been studied by means of labelled lectins using enhanced chemiluminescence detection. The results demonstrated that individual submandibular kallikreins are not heavily glycosylated in rats, but consistently show different patterns of glycosylation. Following digestion of slot-blotted enzymes with peptide-N-glycosidase F (PNGase): binding by lectin fromLens culinaris (Man-directed) was abolished, whilst that of lectin fromMaclura pomifera (Gal1,3GalNAc-directed) persisted (but could be abolished by periodate oxidation and endo--N-acetylgalactosaminidase digestion), revealing that there are O- as well as N-linked sugar chains on the kallikreins; a novel observation for this family of enzymes. The presence of GalNAc in addition to GlcNAc, Fuc, Gal and Man, in sugar chains of rK1 was confirmed by high pH anion exchange chromatography following acid hydrolysis. Different intensities of binding by lectin fromLimax flavus (NeuNAc-directed) suggest that sialylation of individual kallikreins differs, whilst sialidase and PNGase digestions suggest that sialic acid is the terminal residue of some N-linked but not O-linked structures. 相似文献
19.
Henning Ursula Wolf Wolf-Peter Holtzhauer Martin 《Molecular and cellular biochemistry》1996,160(1):41-46
Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells.Exposure of the cells up to 6 g/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (Bmax) to 46.0 ± 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca++ channel by N-glycosylation is an important determinant for the binding of Ca++ antagonists of the DHP-type to the 1 subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded. 相似文献
20.
Georgeen Gaza-Bulseco Ashley Bulseco Chris Chumsae Hongcheng Liu 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,862(1-2):155-160
Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography. 相似文献