BlotGlycoABC™, an Integrated Glycoblotting Technique for Rapid and Large Scale Clinical Glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an “all-in-one” protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC™ bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery. Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an “all-in-one” protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC™ bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery. Large scale and quantitative glycomics is an important and promising approach because the difference in glycan expression between disease and healthy states is expected to be a useful tool for the diagnosis or prognosis of diseases (1Hakomori S. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines.Adv. Exp. Med. Biol. 2001; 491: 369-402Crossref PubMed Scopus (367) Google Scholar). Mass spectrometric methods are now exclusively applied for the structural analysis and sequencing study of carbohydrates (2Zaia J. Mass spectrometry of oligosaccharides.Mass Spectrom. Rev. 2004; 23: 161-227Crossref PubMed Scopus (708) Google Scholar, 3Dell A. Morris H.R. 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A serum glycomics approach to breast cancer biomarkers.Mol. Cell. Proteomics. 2007; 6: 43-55Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), MS is preferred to other analytical methods, such as HPLC and nuclear magnetic resonance spectroscopy, in glycomics. In particular, MALDI-TOF and ESI MS that deliver mild ionization of glycans and high throughput analysis are potential candidates for primary techniques. However, there is no PCR-like glycan amplification technology for glycomics because glycan biosynthetic process is not template-driven and is subject to multiple sequential and competitive enzymatic steps. Although partial peptide fragments detected in proteomics are fully supported by full-length protein/DNA sequence databases, glycomics requires the enrichment of total glycans from highly complicated mixtures such as serum, cells, and tissues. Therefore, one crucial bottleneck in structural and functional glycomics is the tedious and time-consuming multistep process for the purification of trace amounts of glycans. For example, glycans, once released from glycoproteins or glycolipids, are often subjected to fluorescent labeling and purification for detection in HPLC analysis (7Neville D.C. Coquard V. Priestman D.A. te Vruchte D.J. Sillence D.J. Dwek R.A. Platt F.M. Butters T.D. Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.Anal. Biochem. 2004; 331: 275-282Crossref PubMed Scopus (151) Google Scholar, 8Tomiya N. Kurono M. Ishihara H. Tejima S. Endo S. Arata Y. Takahashi N. Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography.Anal. Biochem. 1987; 163: 489-499Crossref PubMed Scopus (104) Google Scholar). 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In some cases, sialic acids are deleted for the simplification or limitation of detection even though important information will be lost. Recently a DNA sequencer has also been utilized for clinical N-glycan profiling (12Callewaert 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 (373) Google Scholar, 13Laroy W. Contreras R. Callewaert N. Glycome mapping on DNA sequencing equipment.Nat. Protoc. 2006; 1: 397-405Crossref PubMed Scopus (150) Google Scholar) in which the above mentioned multistep procedures of purification and derivatization suited for capillary electrophoresis are still required. In general, protocols for the preparation of glycan derivatives vary depending upon the analytical methods and require specialized expertise for step-by-step handling. These technical but crucial problems in the enrichment of glycans make it impossible to achieve reliable and high throughput glycomics (14Morelle W. Canis K. Chirat F. Faid V. Michalski J.C. The use of mass spectrometry for the proteomic analysis of glycosylation.Proteomics. 2006; 6: 3993-4015Crossref PubMed Scopus (180) Google Scholar). Our recent efforts have been directed to the establishment of practical methods for glycan enrichment, namely “glycoblotting” (15Nishimura S.-I. Niikura K. Kurogochi M. Matsushita T. Fumoto M. Hinou H. Kamitani R. Nakagawa H. Deguchi K. Miura N. Monde K. Kondo H. High-throughput protein glycomics: combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry.Angew. Chem. Int. Ed. Engl. 2005; 44: 91-96Crossref Scopus (130) Google Scholar, 16Lohse A. Martins R. Jorgensen M.R. Hindsgaul O. Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures.Angew. Chem. Int. Ed. 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Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.Mol. Cell. Proteomics. 2007; 6: 1437-1445Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The optimized protocol requires only 5 μl of human serum for the quantitative profiling of 30–40 kinds of major glycoforms within 5–8 h (19Miura Y. Shinohara Y. Furukawa J.I. 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 (99) Google Scholar). These attempts clearly indicated the advantage of incorporation of solid support in the facile and efficient manipulation of enriched glycans, yielding higher recovery and reducing complicated operations. We present herein an “all-in-one” solution for automated and high throughput N-glycan enrichment and processing that is greatly beneficial in “real world” clinical glycomics. This system permits the integration of multiple steps such as selective capturing of total glycans, methyl esterification of sialic acids, and fluorescent tagging in a single workflow based on the combined use of a hydrazide-functionalized bead with multiwell filter plate handling. We believe that this de facto standardized technology will allow for large scale clinical glycomics/glycoproteomics and accelerate the discovery of new disease-related biomarkers. The clinical study was approved by the local ethics committee of Hokkaido University Hospital. Informed consent was obtained from all serum donors. Peptide-N-glycosidase F (PNGase F) 1The abbreviations used are: PNGase F, peptide-N-glycosidase F; CDG, congenital disorders of glycosylation; HCC, hepatocellular carcinoma; PCA, principal component analysis; ABCh, N-(2-aminobenzoyl)cysteine hydrazide; CDG-I, type 1 CDG; CDG-II, type 2 CDG; PrEC, prostate epithelial cell. was purchased from Roche Applied Science. 3-Methyl-1-p-tolyltriazene, 8 m borane-pyridine complex, and DTT were from Sigma-Aldrich. BlotGlycoABC™ was prepared as described in the supplemental note and is now commercially available from Sumitomo Bakelite, Co., Tokyo, Japan. Other reagents and solvents were obtained from Wako Pure Chemicals Co., Tokyo, Japan, unless otherwise stated. Sera of congenital disorders of glycosylation (CDG) patients were kindly provided by Prof. H. H. Freeze, Burnham Institute for Medical Research, La Jolla, CA. Whole human serum was treated according to the condition reported previously (20Kita Y. Miura Y. Furukawa J.I. Nakano M. Shinohara Y. Ohno M. Takimoto A. Nishimura S.-I. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.Mol. Cell. Proteomics. 2007; 6: 1437-1445Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) by our group to release reducing N-glycans. Briefly a 10-μl aliquot of serum was diluted 6-fold in 83 mm ammonium bicarbonate containing a detergent and 10 mm DTT. After incubation for 30 min at 60 °C, 1 volume (10 μl) of 123 mm iodoacetamide was added, and the mixture was incubated for an at in the the solution was of and the mixture was incubated for an at After of 2 of PNGase F were to the and the was incubated at The released total and in a mixture were used for glycoblotting by of the μl of was to a of a filter plate The were with 1 m acid and using a μl of whole serum with and PNGase F, to μl of serum, was to the by the of μl of in The plate was incubated at to in a this 30 The plate was with μl of m in mm ammonium bicarbonate by with μl of on-bead methyl esterification was (19Miura Y. Shinohara Y. Furukawa J.I. 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 (99) Google Scholar), the were with μl of 10 mm and The was incubated with μl of mm in for 60 min at 60 The solution was by and the were with and μl) The between the oligosaccharide and the fluorescent on the bead was by incubation with a reducing m according to the method reported previously A. Martins R. Jorgensen M.R. Hindsgaul O. Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures.Angew. Chem. Int. Ed. Engl. 2006; 45: 4167-4172Crossref PubMed Scopus (36) Google Scholar). The was at for 30 min in the After the of the the were with and μl) the μl of mm DTT in 5 mm ammonium bicarbonate was added, and the plate was incubated for 5 min at 60 and an min at The enriched as above were from the in the DTT The in the DTT solution μl) were on a plate with an volume of the a mixture of acid and acid (10 in and to of the The mass were with an in positive ion mode In this a of N-glycan in MALDI-TOF MS were using the were using The with a unless otherwise was because only one of was available for the The of the of glycan was to a of that be used for the differentiation of type 1 and normal we a using an that a mixture of in steps of R.A. and the Rev. Scopus Google Scholar). component analysis was using and we the principal The for the was as of total was used to the difference of disease normal A difference was was the to the between the states and we applied a sequential that a combination of N-glycan based on of a The new method described herein an facile and approach for the enrichment analysis of specialized an we a N-(2-aminobenzoyl)cysteine and used it for the with to hydrazide-functionalized namely the bead the supplemental and supplemental The group with or that are in carbohydrates a reducing of the between the bead and glycans is that reducing be otherwise it be for to a A. Martins R. Jorgensen M.R. Hindsgaul O. Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures.Angew. Chem. Int. Ed. Engl. 2006; 45: 4167-4172Crossref PubMed Scopus (36) Google Scholar). This is to carbohydrates from even in the of and reagents used in proteomics preparation that a of reagents such as Our method quantitative of N-linked type oligosaccharides of sialic The with the quantitative recovery of enriched by with DTT in an The containing neutral and carbohydrates were for the following quantitative analysis using HPLC and some of the MS analysis Although the neutral and sialylated glycans are by the above sialylated oligosaccharides are to MS analysis to stabilization of sialic acids is for the quantitative MS analysis of a esterification of sialic acid residues by was the protocol (19Miura Y. Shinohara Y. Furukawa J.I. 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 (99) Google Scholar). capturing total by of the bead, on-bead methyl esterification of sialic acids and on-bead of the were to mass for neutral and glycoforms positive mode in the new human serum μl) was subjected to the optimized protocol using the An aliquot of the solution to of serum was on a plate and subjected to the MALDI-TOF analysis We and that were in all and to as an are available by PNGase F (20Kita Y. Miura Y. Furukawa J.I. Nakano M. Shinohara Y. Ohno M. Takimoto A. Nishimura S.-I. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.Mol. Cell. Proteomics. 2007; 6: 1437-1445Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) of whole serum it h to N-glycan profiles with a using the bead and MALDI-TOF be that this solid-phase protocol is the of glycoblotting technique in a single We are and a processing suited for the automated glycoblotting using In the glycoblotting protocol based on be to an by combined use with a multiwell filter plate a optimized for the human serum The of the automated protocol was by of the human serum and was by from human serum were in this in a new and profiling of in whole serum glycoproteins is greatly beneficial for the rapid of protein glycosylation by congenital and We our technology on from patients from the disease CDG, a group of human disorders by in protein glycosylation C. Miura Y. N. S. S. R. P. liver disease in a congenital of 2006; PubMed Scopus Google Scholar). of have been M. glycan the basis of congenital disorders of Biol. 2005; PubMed Scopus Google Scholar). from in the of N-glycan or steps of N-glycan the 1 or processing 2 of our glycoblotting technique based on and MALDI-TOF we glycosylation in the whole serum glycoproteins from the analysis was and the in the MALDI-TOF MS were to a of for type 2 patients a in a of glycosylation, we N-glycan profiles such as an of some N-glycan of the other enriched from the N-glycan profiles to the normal subject and However, the also indicated a of the total and for and also supplemental with that of normal clearly that the in results in of whole glycan to some to the in The of using quantitative glycomics to of patients is because the by the of the detected be used for the facile differentiation of patients from normal serum and The results also that a of of N-glycan have been to a differentiation between patients and In addition, we used of total N-glycan profiles to the complex, of all the The principal clearly the including normal results that the of the whole serum N-glycan clearly a of from the other of as from healthy demonstrate the of the present protocol in large scale clinical glycomics, we applied our technique to from multiple patients from use of the glycoblotting protocol based on and MALDI-TOF analysis for rapid and quantitative N-glycan profiling of human serum patients and normal the to the between the disease and we applied a sequential that a combination of N-glycan based on of a a and we the of the that difference between disease and the of N-glycan that from normal with whole serum N-glycan profiling was demonstrated to a tool for diseases that have previously been to or to serum, cells and are also important in clinical glycomics. We applied the enrichment protocol above to cells to total cellular prostate cancer cells and normal human prostate epithelial cells were and whole protein were subjected to bead serum N-glycan total cellular were with a of we have not the glycan profiles using other The results that N-glycan profiles for were detected and that our approach described above for serum glycomics is suited to cellular glycomics The of glycomics is to make the protocol for further high throughput A of that demonstrate in protein glycosylation with disease states have been reported S. of and oligosaccharide in breast of a sialylated J. 2005; Google Scholar, M. J. R. S. analysis of serum glycoproteins in the development of primary hepatocellular 2006; PubMed Scopus Google Scholar, M.V. cancer is with a in the of sialylated and neutral PubMed Scopus Google Scholar, Y. J. Novotny M.V. in the serum to prostate 2007; 6: PubMed Scopus Google Scholar). Our results the potential clinical of quantitative analysis of whole serum N-glycan profiles with MALDI-TOF Recently the of MALDI-TOF MS for the profiling of in serum has been in quantitative and analysis W. C. Michalski J.C. A. P. A. Mass spectrometric approach for of total serum in human to 2006; PubMed Scopus Google Scholar, C. V. Miura Y. V. W. new of type 2007; PubMed Scopus Google Scholar) to the detection of Our method of glycan enrichment and derivatization for the quantitative and high throughput MS analysis in clinical The total were by incorporation of the glycoblotting bead, herein and on-bead methyl The present protocol requires only a aliquot of human whole serum, and the multistep for the derivatization were on a solid in a single including MALDI-TOF the and of of improvements demonstrated in serum glycan preparation and our protocol also be used for glycan enrichment analysis from cellular We demonstrated that the total cellular of human prostate cancer cells and normal cells in a single were with glycoblotting using and MALDI-TOF MS The methods for cellular profiling were the as for serum for the in and the supplemental The results clearly indicated a difference in the N-glycan between cells and normal in and of glycans. We believe that glycomics at the cellular and be greatly by using our Recent on that have that glycan analysis to be to the in some R.A. O. J. J. M. S. of the a congenital Med. 2004; 10: PubMed Scopus Google Scholar, R.A. and the Rev. Scopus Google Scholar). However, our results clearly demonstrated that the of N-glycan in was in expression upon principal component we not multiple for for CDG, discovery of the be through quantitative glycomics at such as the because with other diseases be detected by a simple and we believe that our glycoblotting technique be the approach to use because of and of also be to the technique to other diseases that have been with glycosylation in such as congenital and to the of the analysis in this on the N-glycan profiling by large scale glycomics of patients and normal the approach of a of N-glycan expression that is for We from normal with by of the above N-glycan Therefore, the quantitative of glycoforms have potential clinical information as a and efficient biomarker for human HCC, the information obtained be using other clinical and with of mass were obtained the MALDI-TOF MS analysis because of quantitative methyl esterification of sialic acid which the method to and quantitative profiling of neutral and N-glycans. such other analytical methods, we to to the that the of mass often upon the used the analysis of the of as as of the tagging group and supplemental is that laser applied in system ionization of such as high glycans with multiple sialic acid the present results with our (20Kita Y. Miura Y. Furukawa J.I. Nakano M. Shinohara Y. Ohno M. Takimoto A. Nishimura S.-I. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.Mol. Cell. Proteomics. 2007; 6: 1437-1445Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), the of the fully sialylated glycans was to be that of the This be by a difference in between the high sensitivity (20Kita Y. Miura Y. Furukawa J.I. Nakano M. Shinohara Y. Ohno M. Takimoto A. Nishimura S.-I. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.Mol. Cell. Proteomics. 2007; 6: 1437-1445Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, R. Furukawa J. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. throughput quantitative glycomics and proteomics of and Cell. Proteomics. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and the present in as as a difference in the laser and used in measurement A. A. M. J. laser profiles for Mass Spectrom. 2006; PubMed Scopus Google Scholar). achieve and reliable quantitative analysis in glycomics, we to a new type of that such high glycans with higher ionization We believe that the glycoblotting technique greatly discovery research for new of diseases using multiple serum and cellular the used for solid-phase manipulation of the enriched is suited for the derivatization in DNA sequencer-based the with optimized be used for analytical We H. H. for of patients and and K. Hokkaido for with We also A. Hokkaido for technical with