Quantitative Glycomics of Human Whole Serum Glycoproteins Based on the Standardized Protocol for Liberating N-Glycans
2007; Elsevier BV; Volume: 6; Issue: 8 Linguagem: Inglês
10.1074/mcp.t600063-mcp200
ISSN1535-9484
AutoresYoko Kita, Yoshiaki Miura, Jun‐ichi Furukawa, Mika Nakano, Yasuro Shinohara, Masahiro Ohno, Akio Takimoto, Shin‐Ichiro Nishimura,
Tópico(s)Galectins and Cancer Biology
ResumoGlobal glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis. Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis. Sequencing the human genome and that of various pathogens has opened the door for proteomics, which has dramatically facilitated the search for diagnostic biomarkers. Proteomics approaches have emerged as indispensable tools to identify new disease markers from clinical specimens (1Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer.Lancet. 2002; 359: 572-577Abstract Full Text Full Text PDF PubMed Scopus (2857) Google Scholar, 2Veenstra T.D. Conrads T.P. Hood B.L. Avellino A.M. Ellenbogen R.G. Morrison R.S. Biomarkers: mining the biofluid proteome.Mol. Cell. Proteomics. 2005; 4: 409-418Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In particular, human serum/plasma proteomes are considered the most informative proteomes from a medical/clinical point of view because they likely contain most human proteins as well as proteins derived from some pathogens (3Anderson N.L. Polanski M. Pieper R. Gatlin T. Tirumalai R.S. Conrads T.P. Veenstra T.D. Adkins J.N. Pounds J.G. Fagan R. Lobley A. The human plasma proteome: a nonredundant list developed by combination of four separate sources.Mol. Cell. Proteomics. 2004; 3: 311-326Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar). An additional approach currently in development focuses on serum protein glycomics, the qualitative and quantitative characterization of the gross glycans present in serum (4Dube D.H. Bertozzi C.R. Glycans in cancer and inflammation—potential for therapeutics and diagnostics.Nat. Rev. Drug Discov. 2005; 4: 477-488Crossref PubMed Scopus (1356) Google Scholar). Indeed glycosylation is the most common posttranslational modification of cell surface and extracellular matrix proteins, and most plasma proteins are also thought to be heavily glycosylated. Changes in abundance and alterations in glycan profiles of serum and cell surface proteins have been shown to correlate with progression of cancer and other disease states (5Kim Y.S. Hwang S.Y. Oh S. Sohn H. Kang H.Y. Lee J.H. Cho E.W. Kim J.Y. Yoo J.S. Kim N.S. Kim C.H. Miyoshi E. Taniguchi N. Ko J.H. Identification of target proteins of N-acetylglucosaminyl-transferase V and fucosyltransferase VIII in human gastric tissues by glycomic approach.Proteomics. 2004; 4: 3353-3358Crossref PubMed Scopus (18) Google Scholar, 6Parekh R.B. Dwek R.A. Sutton B.J. Fernandes D.L. Leung A. Stanworth D. Rademacher T.W. Mizuochi T. Taniguchi T. Matsuta K. Takeuchi F. Nagano Y. Miyamoto T. Kobata A. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG.Nature. 1985; 316: 452-457Crossref PubMed Scopus (989) Google Scholar, 7Meezan E. Wu H.C. Black P.H. Robbins P.W. Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by Sephadex chromatography.Biochemistry. 1969; 8: 2518-2524Crossref PubMed Scopus (187) Google Scholar). Recently Callewaert et al. (8Callewaert N. Van Vlierberghe H. Van Hecke A. Laroy W. Delanghe J. Contreras R. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics.Nat. Med. 2004; 10: 429-434Crossref PubMed Scopus (377) Google Scholar) reported the glycomics analysis of 106 patients with chronic liver disorders at various stages of severity and revealed significant alterations in specific N-glycans depending on the presence of cirrhosis. Although prostate-specific antigen tests often suffer from lack of specificity in distinguishing benign prostate hyperplasia from prostate cancer, recent studies indicate that N-glycans of prostate-specific antigen found in prostate cancer differ significantly from those seen in benign prostate hyperplasia and therefore could be a potential indicator leading to improved sensitivity in diagnosing prostate cancer (9Ohyama C. Hosono M. Nitta K. Oh-eda M. Yoshikawa K. Habuchi T. Arai Y. Fukuda M. Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy.Glycobiology. 2004; 14: 671-679Crossref PubMed Scopus (143) Google Scholar, 10Peracaula R. Tabares G. Royle L. Harvey D.J. Dwek R.A. Rudd P.M. de Llorens R. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins.Glycobiology. 2003; 13: 457-470Crossref PubMed Scopus (254) Google Scholar). For glycomics analysis, glycans are often released from protein backbones. Asn-linked type glycans can be cleaved enzymatically by peptide-N-glycosidase F (PNGase F) 1The abbreviations used are: PNGase F, peptide-N-glycosidase F; ALS, acid-labile surfactant; aoWR, Nα-((aminooxy)acetyl)tryptophanylarginine methyl ester; HSD, 2-hydroxyl-3-sulfopropyldodecanoate; IAA, iodoacetamide; MTT, 3-methyl-1-p-tolyltriazene; PA, 2-aminopyridine; PHL, 1-propanesulfonic acid, 2-hydroxy-3-lauramido; PHM, 1-propanesulfonic acid, 2-hydroxy-3-myristamido; RA, rheumatoid arthritis. (peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase, EC 3.5.1.52; previously assigned as EC 3.2.2.18) (11Yosizawa Z. Sato T. Schmid K. Hydrazinolysis of α1-acid glycoprotein.Biochim. Biophys. Acta. 1966; 121: 417-419Crossref PubMed Scopus (92) Google Scholar) or glycoamidase A (EC 3.5.1.52) (12Takasaki S. Mizuochi T. Kobata A. Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides.Methods Enzymol. 1982; 83: 263-268Crossref PubMed Scopus (473) Google Scholar) and chemically by hydrazinolysis (13Takahashi N. Demonstration of a new amidase acting on glycopeptides.Biochem. Biophys. Res. Commun. 1977; 76: 1194-1201Crossref PubMed Scopus (154) Google Scholar, 14Plummer Jr., T.H. Tarentino A.L. Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum.Glycobiology. 1991; 1: 257-263Crossref PubMed Scopus (138) Google Scholar). The former is more preferably used because it yields intact oligosaccharides regardless of size or structure of the substrate carbohydrate moiety and a slightly modified protein in which Asn residues at the site of de-N-glycosylation are converted to Asp, whereas hydrazinolysis causes chemical modification including N-deacetylation of sialic acids and N-acetyl-d-hexosamines such as GlcNAc and GalNAc residues as well as extensive cleavage of polypeptide backbones. However, glycoproteins widely differ in susceptibility to enzymatic digestion because glycosylated sites are often obstructed by secondary and tertiary protein structure. To optimize efficiency of enzymatic release of N-glycans from individual/target glycoproteins, several conditions have been utilized using reducing agents, surfactants, protease treatment, or a combination of pretreatments prior to PNGase F digestion to make glycosylation sites more accessible. Although these procedures are often used to obtain qualitative information on N-glycan structures of specific glycoproteins, there are no standardized conditions allowing highly efficient and reproducible liberation of N-glycans from serum whole glycoprotein. It should be noted that quantitative glycomics is premised on non-biased, highly efficient, and reproducible oligosaccharide liberation from human serum glycoproteins. Therefore, our attention must be directed to establish a standardized procedure for liberating major N-glycans from human whole serum glycoproteins. Using an optimized protocol for quantitative glycomics, we revealed for the first time the absolute concentrations of major N-glycans occurring in human serum whole glycoproteins. 1-Butanol, ammonium bicarbonate, and sodium phosphate buffer were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). 2-Hydroxyl-3-sulfopropyl dodecanoate (HSD), 3-methyl-1-p-tolyltriazene (MTT), and sodium cyanoborohydride were purchased from Aldrich. Trypsin was purchased from Sigma-Aldrich. PNGase F (recombinant) and Pronase were obtained from Hoffmann-La Roche and Calbiochem, respectively. Large scale preparation of PNGase F was carried out according to a previously reported method (14Plummer Jr., T.H. Tarentino A.L. Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum.Glycobiology. 1991; 1: 257-263Crossref PubMed Scopus (138) Google Scholar, 15Tarentino A.L. Gomez C.M. Plummer Jr., T.H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.Biochemistry. 1985; 24: 4665-4671Crossref PubMed Scopus (917) Google Scholar). Briefly Flavobacterium meningosepticum (ATCC33958) was cultured, and the medium was centrifuged and filtered. The extract was concentrated by ultrafiltration, and ammonium sulfate was added to 90%. After centrifugation, the precipitate was resuspended in 0.1 m sodium phosphate buffer (pH 7.0) containing 1 m ammonium sulfate and 1 mm EDTA and then centrifuged. The supernatant was applied to a TSK-butyl-Toyopearl 650 M column, and the PNGase F fraction was collected and lyophilized. Sephadex G-15 resin was obtained from Amersham Biosciences, Bio-Gel P-4 (200–400 mesh) was from Bio-Rad, and a ShimPack HRC-ODS silica column (6.0-mm internal diameter × 150 mm) was from Shimadzu Co. (Kyoto, Japan). 2,5-Dihydroxybenzoic acid, human angiotensin II, bombesin, and adrenocorticotropic hormone 18–39 were from Bruker Daltonics (Bremen, Germany). 2-Aminopyridine, acetonitrile (HPLC/MS grade), methanol (HPLC/MS grade), acetic acid, ammonium acetate, and other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Both forms of Nα-((aminooxy)acetyl)tryptophanylarginine methyl ester (aoWR), aoWR(H) and aoWR(D), were prepared as described previously (16Uematsu R. Furukawa J. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Human sera from two normal subjects (males, 73 and 69 years old, designated as normal sera A and B, respectively) were purchased from Genomics Collaborative Inc. (Cambridge, MA). Human serum was also purchased from Sigma-Aldrich and was designated as normal serum C. A serum sample of a rheumatoid arthritis (RA) patient (female, 52 years old) was obtained from the Department of Orthopaedic Surgery, Hokkaido University School of Medicine (Sapporo, Japan) under the permission of the Commission of Bioethics. 3-Chloro-2-hydroxypropane sulfonic acid sodium salt (1.97 g, 10 mmol) was dissolved in 12.5% aqueous ammonia. The mixture was stirred for 24 h at room temperature, and the solvent was removed under reduced pressure to give 3-amino-2-hydroxypropane sulfonic acid sodium salt in quantitative yield. NaHCO3 (84 mg, 1 mmol) and lauroyl chloride (231 μl, 1 mmol) were added to a solution of amino sulfonic acid (177 mg, 1 mmol) in 10 ml of water, and the mixture was stirred for 2 h at room temperature. The reaction mixture was subjected to chromatography on a column of Wako gel 50C18 (2 cm3) using MeOH as eluant and recrystallized to give PHL (positive ion mode MALDI-TOF m/z for C15H30O5NSNa [M + Na]+, calculated: 382.164; found: 382). 3-Amino-2-hydroxypropane sulfonic acid sodium salt was treated with myristoyl chloride in the same manner as described above to give PHM (positive ion mode MALDI-TOF m/z for C17H34O5NSNa [M + Na]+, calculated: 410.1953; found: 410). Before PNGase F digestion, human serum (20 μl) was pretreated using nine different conditions as follows. In Condition A, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH 7.8) and 26 μl of H2O. In Condition B, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH 7.8), 21 μl of H2O, and 5 μl of trypsin (400 units) followed by incubation at 37 °C for 1 h. Then trypsin was heat denatured at 80 °C for 15 min. In Condition C, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH 7.8), 8 μl of H2O, and 8 μl of 50 mm DTT followed by incubation at 60 °C for 30 min. 5 μl of 135 mm iodoacetamide (IAA) in H2O was added, and the mixture was allowed to stand at room temperature for 1 h. Then 5 μl of trypsin (400 units) was added and incubated at 37 °C for 1 h followed by heat denaturation at 80 °C for 15 min. In Condition D, serum was diluted with an equal volume of 50 mm Tris/HCl buffer (pH 7.8) containing 2% (w/v) SDS and 2% 2-mercaptoethanol and heated to 95 °C for 5 min. Then an equal volume of buffer solution containing 8% (v/v) Triton X-100 was added. In Condition E, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH 7.8), 8 μl of 0.5% acid-labile surfactant (ALS; Waters, Milford, MA) in H2O, 8 μl of H2O, and 8 μl of 50 mm DTT followed by incubation at 55 °C for 45 min. 5 μl of 135 mm IAA in H2O was added, and the mixture was allowed to stand at room temperature for 45 min. Finally 5 μl of H2O was added for PNGase F digestion. In Condition F, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH 7.8), 8 μl of 0.5% HSD in H2O, 8 μl of H2O, and 8 μl of 50 mm DTT followed by incubation at 55 °C for 45 min. 5 μl of 135 mm IAA in H2O was added, and the mixture was allowed to stand at room temperature for 45 min. Finally 5 μl of H2O was added for PNGase F digestion. In Conditions G, H, and I, serum was mixed with 4 μl of 100 mm ammonium bicarbonate (pH ∼7.8); 16 μl of 0.5% HSD (Condition G), 0.05% PHL (Condition H), or 0.005% PHM (Condition I) in H2O; and 8 μl of 50 mm DTT followed by incubation at 55 °C for 45 min. 5 μl of 135 mm IAA in H2O was added, and the mixture was allowed to stand at room temperature for 45 min. Then 5 μl of trypsin (400 units) was added and incubated at 37 °C for 1 h followed by heat denaturation at 80 °C for 15 min. Subsequently all the pretreated serum samples were treated with PNGase F (2 units) at 37 °C for 24 h followed by heat denaturation at 90 °C for 15 min. The final volume of all samples was adjusted to 200 μl with 100 mm ammonium bicarbonate. All sample preparations were performed in triplicate except for Condition D, which was in duplicate. Following enzymatic release of serum N-glycans under the digestion conditions described above, an aliquot of each sample (50 μl) was digested with 20 μg of Pronase, and the mixture was purified by Bio-Gel P-4 column chromatography. Oligosaccharides obtained were reductively aminated with 1.7 m PA and 2.0 m sodium cyanoborohydride at 90 °C for 1 h and then purified on a Sephadex G-15 column using 10 mm ammonium bicarbonate as eluant (17Nakagawa H. Kawamura Y. Kato K. Shimada I. Arata Y. Takahashi N. Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.Anal. Biochem. 1995; 226: 130-138Crossref PubMed Scopus (127) Google Scholar). After removing the solvent, the sample was dissolved in 500 μl of water, and a 5-μl aliquot was injected into the reversed-phase HPLC system. Disialylated biantennary oligosaccharide (A2; NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→2)Manα(1→6)[NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→2)Manα(1→3)]Manα(1→3)Manβ(1→4)GlcNAcβ(1→4)GlcNAc) was prepared by PNGase F digestion of sialylglycopeptide, which was purified from hen egg yolk (18Seko A. Koketsu M. Nishizono M. Enoki Y. Ibrahim H.R. Juneja L.R. Kim M. Yamamoto T. Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.Biochim. Biophys. Acta. 1997; 1335: 23-32Crossref PubMed Scopus (205) Google Scholar). Briefly fresh egg yolk was treated with phenol, and the supernatant was purified by gel filtration (Sephadex G-50 column and Sephadex G-25 column) and chromatographed on an anion exchange column (DEAE-Toyopearl 650 M) and then a cation exchange column (CM-Sephadex C-25). Purified sialylglycopeptide was digested with PNGase F, and then a standard PA-oligosaccharide was prepared with the released sialyloligosaccharide by the procedure noted above. PA-oligosaccharides were applied to an octadecylsilyl silica (ODS, 6 × 150-mm; Shimadzu, Kyoto, Japan) HPLC column. A linear gradient elution was applied at a flow rate of 1.0 ml/min at 55 °C using 10 mm sodium phosphate buffer (pH 3.8) (solvent A) and solvent A containing 0.5% 1-butanol (solvent B) (A/B = 80:20 (0 min), 45:55 (70 min)). Fluorescence was monitored at 400 nm with excitation at 320 nm. Following enzymatic release of serum N-glycans under Condition G, an aliquot of the sample (equivalent to 2 μl of serum) was digested with 2 μg of Pronase, and the mixture was subjected to purification on a Bio-Gel P-4 column. Whole N-glycans obtained were subjected to methyl esterification by treatment with MTT in DMSO-acetonitrile according to previously reported conditions with slight modification (19Miura Y. Shinohara Y. Furukawa J. Nagahori N. Nishimura S.-I. Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry.Chem. Eur. J. 2007; 13: 4797-4804Crossref PubMed Scopus (102) Google Scholar). Briefly lyophilized material (N-glycans) was dissolved in 20 μl of 100 mm HCl, and 480 μl of acetonitrile was added. The sample was then applied onto ∼20 mg of Iatrobeads silica gel (Iatron Laboratories, Inc., Tokyo, Japan) packed in a disposable filter column, Mobicol polypropylene column (1 ml, MoBiTec, Göttingen, Germany), which had been preequilibrated with 1 m acetic acid and acetonitrile. The column was washed with acetonitrile by centrifugation. With the bottom cap in place, 100 μl of 100 mm MTT in a 1:1 mixture of acetonitrile and DMSO was added, and the column was incubated for 1 h at 60 °C. With the bottom cap still in place, 500 μl of acetonitrile was added to the column and briefly mixed. Next the bottom cap was removed. The column was washed with acetonitrile, 2% acetic acid in acetonitrile, and 96% acetonitrile in water, successively. The methyl esterified free oligosaccharides were eluted from the silica gel by 50% aqueous acetonitrile. The recovered oligosaccharides were labeled with aoWR according to a method described previously (16Uematsu R. Furukawa J. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Briefly an aliquot (50 μl) of the eluate (200 μl) was directly mixed with 5 μl of 2 mm aoWR and 50 μl of 2% acetic acid in acetonitrile, and the mixture was heated to 60 °C until the solvent evaporated (∼1 h). Methyl-protected and aoWR-labeled N-glycans were dissolved in 10 μl of water, the mixture was directly mixed with 2,5-dihydroxybenzoic acid (10 mg/ml in 30% acetonitrile) at a 1:10 dilution, and an aliquot (1 μl) was deposited on a stainless steel target plate. MALDI-TOF data were obtained using an Ultraflex time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a LIFT-TOF/TOF facility controlled by FlexControl 2.0 software according to the general procedure reported previously (20Kurogochi M. Nishimura S.-I. Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry.Anal. Chem. 2004; 76: 6097-6101Crossref PubMed Scopus (65) Google Scholar, 21Hato M. Nakagawa H. Kurogochi M. Akama T.O. Marth J.D. Fukuda M.N. Nishimura S.-I. Unusual N-glycan structures in α-mannosidase II/IIx double null embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI-TOF/TOF mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 2146-2157Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). All spectra were obtained using a reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26.3 kV, and a pulsed ion extraction of 160 ns in the positive ion mode. These spectra were the sum of 1,000 laser shots. All peaks were picked by FlexAnalysis 2.0 using the Sophisticated Numerical Annotation Procedure (SNAP) algorithm that fits isotopic patterns to the matching experimental data. The algorithm provides the monoisotopic mass, the intensity and area under the envelope of the isotopic cluster, and the resolution of the peaks in the cluster. Estimation of N-linked type oligosaccharide structures was obtained by input of peak masses into the GlycoMod Tool (Swiss Institute of Bioinformatics) and GlycoSuite (Proteome Systems). Kinetics analysis of PNGase F was carried out at 37 °C in 100 mm ammonium bicarbonate (200 μl), and the reaction was terminated by heating in boiling water. Each velocity was determined at a 30–150 μm N-glycan concentration using serum denatured under Condition G. The amount of released oligosaccharides was determined by reductive amination with PA and HPLC analysis as described above. Initial rates were defined as the amount of product formed after incubation for 60 min, and Kmapp and Vmax values were determined by Sigma Plot, Enzyme Kinetics Module (SYSTAT Software Inc., Chicago, IL). Deglycosylation using PNGase F was characterized by Michaelis-Menten kinetics. Glycoproteins differ widely in susceptibility to PNGase F deglycosylation such that they often require denaturation prior to enzymatic treatment. Glycoproteins are typically denatured by heating in an appropriate detergent (e.g. SDS (22Goodarzi M.T. Turner G.A. Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease.Glycoconj. J. 1998; 15: 469-475Crossref PubMed Scopus (38) Google Scholar, 23Mann A.C. Self C.H. Turner G.A. A general method for the complete deglycosylation of a wide variety of serum glycoproteins using peptide-N-glycosidase-F.Glycosylation Dis. 1994; 1: 253-261Crossref Scopus (13) Google Scholar) or ALS (24Yu Y.Q. Gilar M. Lee P.J. Bouvier E.S. Gebler J.C. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins.Anal. Chem. 2003; 75: 6023-6028Crossref PubMed Scopus (270) Google Scholar)) or by protease (e.g. trypsin or chymotrypsin) pretreatment (17Nakagawa H. Kawamura Y. Kato K. Shimada I. Arata Y. Takahashi N. Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.Anal. Biochem. 1995; 226: 130-138Crossref PubMed Scopus (127) Google Scholar, 25Yu Y.Q. Gilar M. Kaska J. Gebler J.C. A rapid sample preparation method for mass spectrometric characterization of N-linked glycans.Rapid Commun. Mass Spectrom. 2005; 19: 2331-2336Crossref PubMed Scopus (82) Google Scholar, 26Nakano M. Kakehi K. Lee Y.C. Sample clean-up method for analysis of complex-type N-glycans released from glycopeptides.J. Chromatogr. A. 2003; 1005: 13-21Crossref PubMed Scopus (19) Google Scholar) with or without reductive alkylation. ALS, sodium-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxyl]-1-propanesulfonate, is designed to degrade at low pH condition to eliminate surfactant-caused interference with analysis (24Yu Y.Q. Gilar M. Lee P.J. Bouvier E.S. Gebler J.C. Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins.Anal. Chem. 2003; 75: 6023-6028Crossref PubMed Scopus (270) Google Scholar). However, one of the decomposition products contains a ketone group; hence it can seriously interfere with labeling of oligosaccharides required to improve detection sensitivity via reductive amination or hydrazone/oxime formation toward the hemiacetal-reducing terminus. To overcome this difficulty, we sought alternatives with chemical properties similar to ALS and chose to evaluate HSD as a designated surfactant (Fig. 1). A quantitative comparison among different digestion conditions was evaluated by an established reversed-phase HPLC method following pyridylamination of released oligosaccharides. The areas of 14 major peaks in Fig. 2 (major oligosaccharide(s) present in each peak (17Nakagawa H. Kawamura Y. Kato K. Shimada I. Arata Y. Takahashi N. Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.Anal. Biochem. 1995; 226: 130-138Crossref PubMed Scopus (127) Google Scholar, 27Takegawa Y. Deguchi K. Ito S. Yoshioka S. Nakagawa H. Nishimura S.-I. Simultaneous analysis of 2-aminopyridine-derivatized neutral and sialylated oligosaccharides from human serum in the negative-ion mode by sonic spray ionization ion trap mass spectrometry.Anal. Chem. 2005; 77: 2097-2106Crossref PubMed Scopus (38) Google Scholar) are shown in Table I) were used for quantitative analysis.Fig. 2Reversed-phase chromatogram showing separation of PA-derivatized oligosaccharides of PNGase F-treated human serum. The numbers correspond to the structures in Table I. AU, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IMajor oligosaccharides in each peak Fig. 2 Open table in a new tab As shown in Fig. 3a, the total amount of deglycosylated glycans differs significantly depending on the conditions used. When enzyme digestion was performed without denaturation pretreatment (Condition A), the releasing efficiency was significantly low, supporting previous findings that denaturation of substrate before deglycosylation is indispensable for high efficiency release of glycans. Tryptic digestion prior to PNGase F digestion (Condition B) and in combination with reductive alkylation (Condition C) improved efficiencies by ∼88 and ∼127%, respectively, over Condition A. Likewise deglycosylation efficiency was improved following treatment with solubilizing agents combined with reductive alkylation, although improvement di
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