Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) is a powerful tool in proteomics analysis. The optimum conditions for preparing and the performance of a high efficiency polymeric column was compared with micro particle-filled capillary columns, including a totally porous silica C18 column and a HALO® fused core C18 column. Tryptic peptides were used as model compounds for evaluating the performance of three in-house fabricated columns. After optimization, a monolithic capillary column was prepared by the in-situ polymerization of styrene and divinylbenzene (SDVB) within a 50 μm i.d. fused silica capillary using 1-propanol as the porogen. These continuous unitary porous structures are more robust and efficient compared with bead-based columns. Since meter level SDVB column could substantially reduce peptides co-elution and abate the ion suppression, thus permitting the total ion current signal to be significantly enhanced. For the routine identification of peptides, the performances of these three columns were comparable. For glycopeptides, the monolithic SDVB column gave the highest separation efficiency and a total of 20 N-linked glycopeptides could be identified in a tryptic digest of fetuin and bevacizumab (Avastin). The results indicate that an SDVB column possesses great potential for separating hydrophilic peptides. The novel monolithic media described represents a promising addition to the stationary phase used in capillary columns for proteome research. Another part is about glycopeptide analysis. In the development of biosimilar protein drugs, glycan profiling mapping is critical for providing its biosimilarity. The quantitation and structure information of glycans in antibodies or other glycoprotein drugs for pharmaceutical industries is very important. In this study, a sample preparation method using trypsin digestion with RapiGest SF surfactant (Waters) followed by reverse phase nanoLC-MS/MS was compared to the traditional normal phase HPLC-Fluorsecence with 2-AB (2‑aminobenzamide) labeling and the reduced molecular weight analysis. Glycopeptide-based analysis with fast digestion method provides a novel approach for structural analysis of glycans with relative quantitation. The purpose of this research is to evaluate the optimized method for monoclonal antibody (mAb) digested condition for the glycan type determination and their relative abundance of mAb sample by LC-MS/MS. The results indicate that glycopeptide-based analysis used to provide information of the glycans more convenient on mAb’s characterization. In summary, the optimized preparation of styrene-divinylbenzene copolymer column and glycoprotein digestion reaction optimization combined with nanoLC-MS/MS were developed and investigated, which can be beneficial to protein characterization research.

(1) Cunliffe, J. M.; Maloney, T. D. Fused-core particle technology as an alternative to sub-2-microm particles to achieve high separation efficiency with low backpressure. Journal of separation science 2007, 30, 3104-3109.
(2) Salisbury, J. J. Fused-Core Particles: A Practical Alternative to Sub-2 Micron Particles. J Chromatogr Sci 2008, 46, 883-886.
(3) Kirkland, J. J.; Schuster, S. A.; Johnson, W. L.; Boyes, B. E. Fused-core particle technology in high-performance liquid chromatography: An overview. Journal of Pharmaceutical Analysis 2013, 3, 303-312.
(4) Belenkii, B. G. Monolithic stationary phases: Yesterday, today, and tomorrow. Russian Journal of Bioorganic Chemistry 2006, 32, 323-332.
(5) Bakry, R.; Huck, C. W.; Bonn, G. K. Recent applications of organic monoliths in capillary liquid chromatographic separation of biomolecules. J Chromatogr Sci 2009, 47, 418-431.
(6) Svec, F.; Frechet, J. M. J. Continuous rods of macroporous polymer as high-performance liquid chromatography separation media. Analytical Chemistry 1992, 64, 820-822.
(7) Monolithic Materials: Preparation, Properties and Applications; Elsevier Science, 2003.
(8) Urban, J.; Jandera, P. Polymethacrylate monolithic columns for capillary liquid chromatography. Journal of separation science 2008, 31, 2521-2540.
(9) Gusev, I.; Huang, X.; Horváth, C. Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography. Journal of Chromatography A 1999, 855, 273-290.
(10) Buchmeiser, M. R. Polymeric monolithic materials: Syntheses, properties, functionalization and applications. Polymer 2007, 48, 2187-2198.
(11) Duan, Y.; Liu, H.; Li, J.; Ma, J.; Gu, Y.; Yan, C.; Yang, G. Preparation and Evaluation of a Porous P(NIPAAm-MAA-EDMA) Monolithic Column for HPLC. Chromatographia 2011, 75, 87-93.
(12) Okanda, F. M.; El Rassi, Z. Affinity monolithic capillary columns for glycomics/proteomics: 1. Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity capillary electrochromatography and affinity nano-liquid chromatography in either a single column or columns coupled in series. Electrophoresis 2006, 27, 1020-1030.
(13) Huck, C. W.; Bonn, G. K. Poly(Styrene-Divinylbenzene) Based Media for Liquid Chromatography. Chemical Engineering & Technology 2005, 28, 1457-1472.
(14) Ivanov, A. R.; Zang, L.; Karger, B. L. Low-Attomole Electrospray Ionization MS and MS/MS Analysis of Protein Tryptic Digests Using 20-μm-i.d. Polystyrene−Divinylbenzene Monolithic Capillary Columns. Analytical chemistry 2003, 75, 5306-5316.
(15) Huber, C. G.; Walcher, W.; Timperio, A. M.; Troiani, S.; Porceddu, A.; Zolla, L. Multidimensional proteomic analysis of photosynthetic membrane proteins by liquid extraction-ultracentrifugation-liquid chromatography-mass spectrometry. Proteomics 2004, 4, 3909-3920.
(16) Huck, C. W.; Bakry, R.; Bonn, G. K. Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High-Performance Liquid Chromatography-Electrospray Ionisation Mass Spectrometry. Engineering in Life Sciences 2005, 5, 431-435.
(17) Jochum, M.; Bakry, R.; Wartusch, I.; Huck, C. W.; Engelhardt, H.; Bonn, G. K. Analysis of carbohydrates using different quaternized polystyrene-divinylbenzene particles and pulsed amperometric detection. Chromatographia 2002, 56, 263-268.
(18) Corradini, C.; Corradini, D.; Huber, C. G.; Bonn, G. K. Synthesis of a polymeric-based stationary phase for carbohydrate separation by high-pH anion-exchange chromatography with pulsed amperometric detection. Journal of Chromatography A 1994, 685, 213-220.
(19) Zha, W.; Chen, D.; Fei, W. The Hplc Separation of Fullerenes with 1-Methylnaphthalene Modified Psdvb Resin. Journal of Liquid Chromatography & Related Technologies 1999, 22, 2443-2453.
(20) Ali, I.; Gaitonde, V. D.; Aboul-Enein, H. Y. Monolithic Silica Stationary Phases in Liquid Chromatography. Journal of Chromatographic Science 2009, 47, 432-442.
(21) Beneito-Cambra, M.; Herrero-Martinez, J. M.; Ramis-Ramos, G.; Lindner, W.; Lammerhofer, M. Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products. Journal of chromatography. A 2011, 1218, 7275-7280.
(22) Santos, J. C. S. d.; Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; Fernandez-Lafuente, R. Importance of the Support Properties for Immobilization or Purification of Enzymes. ChemCatChem 2015, 7, 2413-2432.
(23) Ma, J.; Zhang, L.; Liang, Z.; Zhang, W.; Zhang, Y. Monolith-based immobilized enzyme reactors: recent developments and applications for proteome analysis. Journal of separation science 2007, 30, 3050-3059.
(24) Wei, F.; Feng, Y.-Q. Methods of sample preparation for determination of veterinary residues in food matrices by porous monolith microextraction-based techniques. Analytical Methods 2011, 3, 1246.
(25) Luo, Q.; Shen, Y.; Hixson, K. K.; Zhao, R.; Yang, F.; Moore, R. J.; Mottaz, H. M.; Smith, R. D. Preparation of 20-microm-i.d. silica-based monolithic columns and their performance for proteomics analyses. Anal Chem 2005, 77, 5028-5035.
(26) Xie, C.; Ye, M.; Jiang, X.; Jin, W.; Zou, H. Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis. Molecular & cellular proteomics : MCP 2006, 5, 454-461.
(27) Miyamoto, K.; Hara, T.; Kobayashi, H.; Morisaka, H.; Tokuda, D.; Horie, K.; Koduki, K.; Makino, S.; Nunez, O.; Yang, C.; Kawabe, T.; Ikegami, T.; Takubo, H.; Ishihama, Y.; Tanaka, N. High-efficiency liquid chromatographic separation utilizing long monolithic silica capillary columns. Anal Chem 2008, 80, 8741-8750.
(28) Horie, K.; Kamakura, T.; Ikegami, T.; Wakabayashi, M.; Kato, T.; Tanaka, N.; Ishihama, Y. Hydrophilic interaction chromatography using a meter-scale monolithic silica capillary column for proteomics LC-MS. Anal Chem 2014, 86, 3817-3824.
(29) Iwasaki, M.; Miwa, S.; Ikegami, T.; Tomita, M.; Tanaka, N.; Ishihama, Y. One-dimensional capillary liquid chromatographic separation coupled with tandem mass spectrometry unveils the Escherichia coli proteome on a microarray scale. Anal Chem 2010, 82, 2616-2620.
(30) Yi, E. C.; Lee, H.; Aebersold, R.; Goodlett, D. R. A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. Rapid communications in mass spectrometry : RCM 2003, 17, 2093-2098.
(31) Ko, C. H.; Cheng, C. F.; Lai, C. P.; Tzu, T. H.; Chiu, C. W.; Lin, M. W.; Wu, S. Y.; Sun, C. Y.; Tseng, H. W.; Wang, C. C.; Kuo, Z. K.; Wang, L. M.; Chen, S. F. Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry. Journal of proteome research 2013, 12, 3573-3585.
(32) Svec, F.; Frechet, J. M. J. Temperature, a Simple and Efficient Tool for the Control of Pore Size Distribution in Macroporous Polymers. Macromolecules 1995, 28, 7580-7582.
(33) Miheli, I.; Koloini, T.; Podgornik, A. Temperature distribution effects during polymerization of methacrylate-based monoliths. Journal of Applied Polymer Science 2003, 87, 2326-2334.
(34) Kurganov, A. Monolithic column in gas chromatography. Anal Chim Acta 2013, 775, 25-40.
(35) Xie, S.; Svec, F.; Fréchet, J. M. J. Rigid porous polyacrylamide-based monolithic columns containing butyl methacrylate as a separation medium for the rapid hydrophobic interaction chromatography of proteins. Journal of Chromatography A 1997, 775, 65-72.
(36) Kittelson, D. B. Engines and nanoparticles. Journal of Aerosol Science 1998, 29, 575-588.
(37) Bezerra, R. M.; Neto, D. M. A.; Galvão, W. S.; Rios, N. S.; Carvalho, A. C. L. d. M.; Correa, M. A.; Bohn, F.; Fernandez-Lafuente, R.; Fechine, P. B. A.; de Mattos, M. C.; dos Santos, J. C. S.; Gonçalves, L. R. B. Design of a lipase-nano particle biocatalysts and its use in the kinetic resolution of medicament precursors. Biochemical Engineering Journal 2017, 125, 104-115.
(38) Tanaka, N.; Nagayama, H.; Kobayashi, H.; Ikegami, T.; Hosoya, K.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Cabrera, K.; Lubda, D. Monolithic Silica Columns for HPLC, Micro-HPLC, and CEC. Journal of High Resolution Chromatography 2000, 23, 111-116.
(39) Ali, I.; Gaitonde, V. D.; Grahn, A. Halo Columns: New Generation Technology for High Speed Liquid Chromatography. J Chromatogr Sci 2010, 48, 386-394.
(40) Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 1982, 157, 105-132.
(41) Gilar, M.; Yu, Y. Q.; Ahn, J.; Xie, H.; Han, H.; Ying, W.; Qian, X. Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry. Analytical biochemistry 2011, 417, 80-88.
(42) Kobata, A.; Amano, J. Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours. Immunol Cell Biol 2005, 83, 429-439.
(43) Dube, D. H.; Bertozzi, C. R. Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 2005, 4, 477-488.
(44) Arnold, J. N.; Saldova, R.; Galligan, M. C.; Murphy, T. B.; Mimura-Kimura, Y.; Telford, J. E.; Godwin, A. K.; Rudd, P. M. Novel glycan biomarkers for the detection of lung cancer. Journal of proteome research 2011, 10, 1755-1764.
(45) Zhang, J.; Wang, D. I. C. Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-γ from Chinese hamster ovary cell culture by hydrophilic interaction chromatography. Journal of Chromatography B: Biomedical Sciences and Applications 1998, 712, 73-82.
(46) Kunkel, J. P.; Jan, D. C.; Butler, M.; Jamieson, J. C. Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors. Biotechnol Prog 2000, 16, 462-470.
(47) Delorme, E.; Lorenzini, T.; Giffin, J.; Martin, F.; Jacobsen, F.; Boone, T.; Elliott, S. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 2002, 31, 9871-9876.
(48) Kristic, J.; Vuckovic, F.; Menni, C.; Klaric, L.; Keser, T.; Beceheli, I.; Pucic-Bakovic, M.; Novokmet, M.; Mangino, M.; Thaqi, K.; Rudan, P.; Novokmet, N.; Sarac, J.; Missoni, S.; Kolcic, I.; Polasek, O.; Rudan, I.; Campbell, H.; Hayward, C.; Aulchenko, Y.; Valdes, A.; Wilson, J. F.; Gornik, O.; Primorac, D.; Zoldos, V.; Spector, T.; Lauc, G. Glycans are a novel biomarker of chronological and biological ages. J Gerontol A Biol Sci Med Sci 2014, 69, 779-789.
(49) Dekkers, G.; Plomp, R.; Koeleman, C. A.; Visser, R.; von Horsten, H. H.; Sandig, V.; Rispens, T.; Wuhrer, M.; Vidarsson, G. Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans. Sci Rep 2016, 6, 36964.
(50) Saint-Jore-Dupas, C.; Faye, L.; Gomord, V. From planta to pharma with glycosylation in the toolbox. Trends Biotechnol 2007, 25, 317-323.
(51) Reusch, D.; Tejada, M. L. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015, 25, 1325-1334.
(52) Jefferis, R. Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 2009, 8, 226-234.
(53) De Muynck, B.; Navarre, C.; Boutry, M. Production of antibodies in plants: status after twenty years. Plant Biotechnol J 2010, 8, 529-563.
(54) Kabbinavar, F.; Hurwitz, H. I.; Fehrenbacher, L.; Meropol, N. J.; Novotny, W. F.; Lieberman, G.; Griffing, S.; Bergsland, E. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003, 21, 60-65.
(55) Presta, L. G.; Chen, H.; Connor, S. J.; Chisholm, V.; Meng, Y. G.; Krummen, L.; Winkler, M.; Ferrara, N. Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal Antibody for the Therapy of Solid Tumors and Other Disorders. Cancer Research 1997, 57, 4593.
(56) Hurwitz, H.; Fehrenbacher, L.; Novotny, W.; Cartwright, T.; Hainsworth, J.; Heim, W.; Berlin, J.; Baron, A.; Griffing, S.; Holmgren, E.; Ferrara, N.; Fyfe, G.; Rogers, B.; Ross, R.; Kabbinavar, F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004, 350, 2335-2342.
(57) Jefferis, R. Posttranslational Modifications and the Immunogenicity of Biotherapeutics. J Immunol Res 2016, 2016, 5358272.
(58) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianferani, S. Characterization of therapeutic antibodies and related products. Analytical chemistry 2013, 85, 715-736.
(59) Wuhrer, M.; Deelder, A. M.; Hokke, C. H. Protein glycosylation analysis by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2005, 825, 124-133.
(60) Gadgil, H. S.; Bondarenko, P. V.; Pipes, G. D.; Dillon, T. M.; Banks, D.; Abel, J.; Kleemann, G. R.; Treuheit, M. J. Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis. Anal Biochem 2006, 355, 165-174.
(61) Sinha, S.; Pipes, G.; Topp, E. M.; Bondarenko, P. V.; Treuheit, M. J.; Gadgil, H. S. Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs. J Am Soc Mass Spectrom 2008, 19, 1643-1654.
(62) Gadgil, H. S.; Pipes, G. D.; Dillon, T. M.; Treuheit, M. J.; Bondarenko, P. V. Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies. J Am Soc Mass Spectrom 2006, 17, 867-872.
(63) Gadgil, H. S.; Bondarenko, P. V.; Pipes, G.; Rehder, D.; McAuley, A.; Perico, N.; Dillon, T.; Ricci, M.; Treuheit, M. The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations. J Pharm Sci 2007, 96, 2607-2621.
(64) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem 1995, 230, 229-238.
(65) Chandler, K. B.; Pompach, P.; Goldman, R.; Edwards, N. Exploring site-specific N-glycosylation microheterogeneity of haptoglobin using glycopeptide CID tandem mass spectra and glycan database search. Journal of proteome research 2013, 12, 3652-3666.
(66) Desaire, H. Glycopeptide analysis, recent developments and applications. Mol Cell Proteomics 2013, 12, 893-901.
(67) Kaji, H.; Yamauchi, Y.; Takahashi, N.; Isobe, T. Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging. Nat Protoc 2006, 1, 3019-3027.
(68) Zhu, Z.; Hua, D.; Clark, D. F.; Go, E. P.; Desaire, H. GlycoPep Detector: a tool for assigning mass spectrometry data of N-linked glycopeptides on the basis of their electron transfer dissociation spectra. Analytical chemistry 2013, 85, 5023-5032.
(69) Nomura, E.; Katsuta, K.; Ueda, T.; Toriyama, M.; Mori, T.; Inagaki, N. Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping. J Mass Spectrom 2004, 39, 202-207.
(70) Ercan, A.; Kohrt, W. M.; Cui, J.; Deane, K. D.; Pezer, M.; Yu, E. W.; Hausmann, J. S.; Campbell, H.; Kaiser, U. B.; Rudd, P. M.; Lauc, G.; Wilson, J. F.; Finkelstein, J. S.; Nigrovic, P. A. Estrogens regulate glycosylation of IgG in women and men. JCI Insight 2017, 2, e89703.
(71) Mauko, L.; Nordborg, A.; Hutchinson, J. P.; Lacher, N. A.; Hilder, E. F.; Haddad, P. R. Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection. Anal Biochem 2011, 408, 235-241.