Assignment of Core versus Antenna Fucosylation Types in Protein N

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Rapid Assignment of Core versus Antenna Fucosylation Types in Protein Nglycosylation via Procainamide Labeling and Tandem Mass Spectrometry Charles Chuks Nwosu, Hoi Kei (Natalie) Yau, and Steven Becht Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 15 May 2015 Downloaded from http://pubs.acs.org on May 16, 2015

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Analytical Chemistry


Rapid Assignment of Core versus Antenna Fucosylation Types in Protein N-glycosylation via Procainamide Labeling and Tandem Mass Spectrometry

Charles Nwosu1*, Hoi Kei (Natalie) Yau1 and Steven Becht1

1. Pharmaceutical Product Development, 8551 Research Way, Middleton, Wisconsin, USA.

* To

whom correspondence should be addressed:

Charles C. Nwosu, email address: [email protected]; Tel: +1 530 220 5356

1

ACS Paragon Plus Environment


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Abstract

Fucosylation is an important feature of protein N-glycosylation as it has been reported to influence the efficacy of therapeutic proteins and as a potential disease biomarker. A common approach for characterizing protein N-glycans is to analyze the native glycans via tandem mass spectrometry (MS). However, tandem MS analysis of native N-glycans typically results in proton migration, which in turn leads to fucose residue migration from the glycan core to the antenna and vice versa. This phenomenon ultimately leads to ambiguous assignment of N-glycan fucosylation. Although the use of specific fucosidases has been successfully employed for assigning fucosylation, such strategies are often too cumbersome, expensive and time consuming for routine N-glycan analysis. As an alternative, we explore the influence of labeling N-glycans with procainamide hydrochloride to inhibit fucose migration during tandem MS analysis. The labeled N-glycan pool was separated and analyzed using ultra-performance liquid chromatography and a hydrophobic interaction liquid chromatography column coupled to a quadrupole time-of-flight mass spectrometer (UPLC-HILIC-QToF-MS). The observation of the m/z 587.3 +

core fucose diagnostic peak corresponding to [GlcNAc + Fucose + Procainamide + H] in the tandem MS data of fucosylated N-glycans rapidly verifies core fucosylation while its absence signifies antennae fucosylation. This unique approach is here validated with human IgG (for core fucosylation) and human alpha-1-acid-glycoprotein (for antenna fucosylation). We further present a useful application toward the rapid verification of fucosylation types in a therapeutic protein (Rituximab).

2


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Introduction

Glycosylation is a common and important form of post-translational modification (PTM) in proteins involving the enzymatic attachment of O-glycans to serine/threonine or N-glycans to asparagine side 1

chains of proteins. Glycosylation plays vital roles in protein-folding recognition activity.

4,8

3,4

2,3

4

, protein stability , cell-cell

5

, immune system , protection of proteins from proteolytic degradation

4,6,7

and protein

Unlike proteomic studies, where there is a template for the peptide backbone, glycomics

requires more rigorous analyses as glycans lack a template for their structures. N-glycans are particularly 9

complicated by their monosaccharide compositions, branching, linkage and connectivity. N-glycans are classified as high mannose, complex and hybrid types depending on their monosaccharide composition and their branching. Glycomics approaches toward profiling N-glycans typically employ the release of the glycans via peptide N-glycosidase F (PNGase F) treatment prior to mass spectrometric (MS) analysis.

10,11

The use of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) is becoming the instrument of choice for N-glycan analysis as LC-MS enables the separation of glycans, thus minimizing suppression effects during ionization.

12

Fucosylation is a particularly important structural feature of protein N-glycans. For example, fucosylation has been reported as a potential marker for diseases such as cancer.

13-15

Additionally,

fucosylation in therapeutic proteins such as monoclonal antibodies (mAbs) has been reported to be significantly relevant to a drug’s efficacy and safety.

16-36

As a quality control requirement in the

biopharmaceutical industry, it is imperative to verify that the fucosylation types and levels in therapeutic proteins are consistent across manufactured batches and in the comparison of biosimilars to their innovator drugs. There are limited reports on the characterization of N-glycan fucosylation types. This is partly due to the dearth of analytical techniques capable of distinguishing between core and antenna fucosylation in N-glycans. Also, issues related to fucose migration during routine tandem MS analysis of native N-glycans

37,38

have further hampered such characterization as such migration often leads to

ambiguous assignments. A few studies have been reported with significant successes toward characterizing protein fucosylation. The use of certain fucosidases to hydrolyze specific fucose residues depending on their 3



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linkage has been reported.

39,40

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While an alpha-1,6 fucosidase specifically hydrolyzes a core fucose

residue, the alpha-1,3 and alpha-1,4 fucosidases hydrolyze antenna fucose residues depending on the fucose residue linkage on the glycan’s antenna. In such studies, the residual glycan mass (detected via mass spectrometry) following treatment with either the core or antenna fucosidase reveals the actual location of the fucose residue. While this approach has been successful, it is expensive, time consuming and often times leads to loss of sample due to the multiple clean-up steps involved. Another common approach toward verifying fucose location in N-glycans is via permethylation experiments.

37

Here, the

released N-glycans are further permethylated prior to tandem MS analysis. Permethylation is useful as it precludes fucose migration during tandem MS analysis.

37

Unfortunately, permethylation is yet to be

extensively adopted for routine N-glycan analysis in the pharmaceutical industry as the analysis of 2-AB labeled N-glycans is still the preferred technique. Klapoetke et. al. recently evaluated procainamide labeling as a better alternative over traditional native or 2-AB labeling of N-glycans toward improved MS and fluorescent detections.

41

Interestingly, the

study reports that the procainamide glycan derivatives generated fluorescent and MS signals that were 10-50 fold improved over the native or 2-AB glycan derivatives. The current study further evaluates the influence of procainamide labeling on the tandem MS analysis of N-glycans toward the unambiguous characterization of fucosylation types in proteins. We show for the first time a strategy that characterizes fucose types based on the presence or absence of a core fucose diagnostic peak in the tandem MS data of procainamide labeled N-glycans. The core fucose diagnostic peak described in this study is the m/z +

587.3 peak that corresponds to [GlcNAc + Fucose + Procainamide + H] . The described strategy is particularly unique in that it provides a platform for the rapid identification of fucosylation types during routine N-glycan analysis via a simple extracted ion chromatogram (XIC) search of the m/z 587.3 core fucose diagnostic peak in the tandem MS data of N-glycans. The m/z of interest, m/z 587.3 in this case, is entered into MassLynx® mass search function, and the m/z of interest is extracted from the total ion chromatogram. A separate chromatogram, XIC, is generated by MassLynx® algorithm to display the m/z of interest. This construction of an XIC is an easy and fast method of data processing. This unique approach is validated with human IgG (for core fucosylation) (for antenna fucosylation)

43

17,42

and human alpha-1-acid-glycoprotein

. Further application is presented for the rapid verification of fucosylation 4


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types in a therapeutic protein (Rituximab). In all, procainamide labeling of N-glycans is a viable alternative to traditional N-glycan analysis strategies as it significantly improves MS sensitivity and enables the rapid characterization of fucosylation types.

Materials and Methods

Materials and chemicals All the reagents were of analytical grade unless stated otherwise. Purified water was obtained from an in-house Milli-Q system. HPLC grade acetonitrile (ACN) was used. PNGase F, Human IgG and the solid phase extraction (SPE) manifold were purchased from Prozyme (Hayward, California). Alpha-1acid-glycoprotein, procainamide hydrochloride, sodium cyanoborohydride, Tris-HCl (pH 8.0) buffer, dimethyl sulfoxide (DMSO) and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, ®

Missouri). Rituximab (Rituxan ) was from Genentech (South San Francisco, California). HILIC µElution plate was purchased from Waters Corporation (Milford, Massachusetts). N-glycan Release N-glycans were released from each protein by incubating approximately 200 µg of each protein o

with 10 µL of 50 mM Tris-HCl buffer (pH 8.0) and 10 µL of PNGase F for 18 hours at 37 C. The digested samples were completely dried in a SpeedVac prior to procainamide labeling. Procainamide Labeling Procainamide labeling reagent was prepared by dissolving 10 mg of procainamide hydrochloride and 6 mg of sodium cyanoborohydride in a 100 µL mixture of dimethyl sulfoxide (DMSO) / acetic acid (7:3, v/v). 10 µL of procainamide labeling reagent was then used to reconstitute each dried o

deglycosylated sample and incubated for 3 hours at 65 C. Glycan Purification Each labeled sample was reconstituted in 200 µL of 95% ACN in water and purified on a HILIC µElution plate. Briefly, each µElution plate well was conditioned under vacuum with 200 µL of purified 5



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water followed with 600 µL of 95% acetonitrile (ACN) in water. Each reconstituted sample was loaded onto individual conditioned plate wells and allowed to drain slowly by vacuum. Each plate well containing the captured glycan sample was rinsed with 200 µL of 95% ACN in water while the rinse solutions were allowed to drain under vacuum. The purified N-glycan samples were each eluted from the plate wells with 50 µL of 20% ACN in water. The eluted glycan samples were each diluted with 150 µL of 0.1% formic acid in ACN and mixed well by mild vortexing. UPLC Instrument and Conditions Profiling of the N-glycan samples was performed on a Waters Acquity UPLC

®

system with

fluorescence (FL) detector. Liquid chromatographic separations were achieved using an Acquity UPLC

®

BEH glycan column (2.1 mm x 100 mm, 1.7 µm, Waters, Milford, Massachusetts) in a column heater set o

at 60 C. The glycans were eluted using a gradient program that included 50 mM ammonium formate in water, pH 4.4 (Mobile Phase A) and 100% ACN (Mobile Phase B). Gradient used was: 20% A (0.0 minutes); 20 to 40% A (0.0 - 46.5 minutes); 40 to 100% A (46.5 – 48.0 minutes); 100% A (48.0 – 49.0 minutes), 100 to 20% A (49.0 – 58.0 minutes) and 20% A (58.0 – 63.0 minutes). The flow rate was 0.5 mL/min with an injection volume of 20 µL, full loop. The excitation and emission wavelengths were 305 ®

nm and 360 nm, respectively. Data acquisition and analysis were performed by using Empower2 from Waters (Milford, Massachusetts) for UPLC-FL data. ESI-QTOF Instrument and Conditions Glycan mass measurement was achieved using a Waters QToF Premier

TM

mass spectrometer

equipped with an electrospray source and lock spray run in positive mode (ES+). MS data was acquired in MS scan mode. Data acquisition and analysis (including the construction of the XICs) were performed ®

using MassLynx (Version 4.1) from Waters (Milford, Massachusetts). Mass spectrometer settings for MS o

o

analyses were as follows: 2.8 KV capillary voltage, 25.0 V cone voltage, 110 C source temperature, 250

e

C desolvation temperature, 100-4000 m/z scan range. The MSMS mass spectra were obtained in MS

mode, and a collision energy ramp from 50 eV to 80 eV was applied to selected precursors. Although the collision energy used in this study was not formally optimized, the ramp proposed is based on previous

6


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experience with the Waters QToF Premier MS instrument

44

where extensive fragmentations were

observed in the analysis of N-glycans (including the important Y-ions needed for this method).

Results and Discussion

Human IgG N-glycan Fucosylation Assignment Human IgG is one of the most abundant proteins in human serum. It is N-glycosylated with most of its glycans containing a single fucose residue. Human IgG is known to be exclusively core fucosylated 17,42

and serves as a good example to verify the described technique with respect to identifying core

fucosylation. Figure 1A represents the fluorescent chromatogram of procainamide labeled human IgG Nglycans, while Figure 1B represents its corresponding tandem MS total ion chromatogram (TIC) data. The human IgG N-glycan peak labels shown in Figure 1A are based on their observed masses and monosaccharide compositions as detailed in Table 1. It is imperative to note that all the fucosylated human IgG N-glycans in Figure 1B are denoted by an asterisk (*) on the corresponding peaks. To verify that the fucosylated N-glycan species observed in human IgG are all core fucosylated, the extracted ion chromatogram (XIC) of m/z (587.30 ± 0.01) was constructed from Figure 1B. The m/z 587.3 XIC profile is +

represented in Figure 1C. The m/z 587.3 corresponds to [GlcNAc + Fucose + Procainamide + H] thus representing a fragment ion uniquely composed of the procainamide tag and the core fucose residue all bound to the protein-connecting GlcNAc residue. As expected, all the human IgG fucosylated N-glycan peaks observed in Figure 1A and 1B have a corresponding m/z 587.3 peak in Figure 1C. For example, there are XIC peaks for the m/z 587.3 corresponding to the elution profile of the G0F, G1F and G2F human IgG N-glycan peaks observed in Figure 1A and 1B. Also, to verify that the m/z 587.3 peak profile is exclusively associated with core fucosylated N-glycan species, there was no m/z 587.3 peak observed in the elution profile corresponding to the G0, G1 and G2 human IgG N-glycan peaks observed in Figure 1A and 1B. In all, these results show that the m/z 587.3 XIC search is a rapid and confident means of verifying the unique fucosylation type(s) contained in protein N-glycosylation. It is also imperative to note that the m/z 587.3 fragment peak

7



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is a universal core fucose diagnostic peak in that it was observed in both neutral and sialylated N-glycan species containing the core fucose residues as observed in the human IgG N-glycan analysis example. MSMS Verification of Human IgG N-glycan Assignment To verify the fucose type assignments made in Figure 1C, the MSMS data for the individual human IgG N-glycan species were evaluated. Figure 2A and 2B represent the MSMS spectra of two human IgG procainamide labeled N-glycans with m/z 1844.775 and m/z 1860.767, respectively. While the N-glycan represented in Figure 2A corresponds to the G1F human IgG N-glycan: [4Hex + 4GlcNAc + +1

1Fuc + Procainamide + H] , the G2 human IgG N-glycan: [5Hex + 4GlcNAc + Procainamide + H]

+1

is

represented in Figure 2B. The core fucose diagnostic peak of m/z 587.3 was observed in Figure 2A indicating that the G1F glycan peak in human IgG is core fucosylated. However, no m/z 587.3 peak was observed in Figure 2B revealing that the G2 glycan peak in human IgG either lacks a fucose residue or is antenna fucosylated. The former is the case for the G2 peak based on the glycan composition assignment represented in Table 1 (per accurate mass measurement). As observed in both examples (Figure 2A and 2B), the majority of the fragment ions include the y-type ions

45,46

with the procainamide

tag and the fucose residue (where applicable) still intact and connected to the core GlcNAc residue. For both examples, extensive glycan fragmentation revealed detailed monosaccharide compositions thus confirming the assignments represented in Table 1. In Figure 2A, it is important to note that the observed +

m/z 790.4 corresponding in mass to [2GlcNAc + Fucose + Procainamide + H] is also a core fucose diagnostic ion. However, the construction of the XICs in this article is based on m/z 587.3 due to its stronger MS signal when compared to the signal from m/z 790.4. This observation was consistent in all data evaluated. Human Alpha-1-Acid Glycoprotein N-glycan Fucosylation Assignment Like human IgG, human alpha-1-acid glycoprotein (AGP) is one of the most abundant glycoproteins in human serum. It has been reported to contain about 45% N-glycosylation with the glycan 43

chains being predominantly those of the complex-type.

The complexity of the AGP N-glycosylation is

predominantly due to its branching, sialylation and fucose residue linkages. AGP is also a good example to verify the proposed technique as it has been confirmed and reported to exclusively contain antenna 8


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fucosylation.

43

Figure 3A represents the fluorescent chromatogram of procainamide labeled AGP N-

glycans while Figure 3B represents its corresponding tandem MS total ion chromatogram (TIC) data. The AGP N-glycan peak labels shown in Figure 3A are based on their observed masses and monosaccharide compositions as detailed in Table 2. All the fucosylated AGP N-glycans in Figure 3B are denoted by an asterisk (*) on the corresponding peaks. To verify that the fucosylated N-glycan species observed in AGP are all antenna fucosylated, the XIC of m/z (587.3 ± 0.01) was constructed from Figure 3B. The m/z 587.3 XIC profile is represented in Figure 3C. The m/z 587.3 peak corresponds to the core fucosylated N-glycan fragment ion, hence no peak is expected in the m/z 587.3 XIC profile of AGP as it is exclusively antenna fucosylated.

43

As expected, no

significant peak was observed in the m/z 587.3 XIC profile of AGP as shown in Figure 3C. Particularly, it is important to note that the G1FS1-N, G2FS2 and G3FS2 peaks observed in AGP (Figure 3A and 3B) each contain one fucose residue. However, there was no significant m/z 587.3 XIC peak corresponding to any of these peaks in Figure 3C. These data confirm that these three AGP fucosylated N-glycans are all antenna fucosylated. Again, the results from these data show that the described technique is a rapid means of verifying fucosylation types in N-glycans. MSMS Verification of Alpha-1-Acid Glycoprotein N-glycan Assignment The MSMS data of the AGP N-glycan peaks were further evaluated to confirm the assignments made in Table 2 and to verify the fucose connection. Figure 4A and 4B represent the MSMS spectra for two AGP procainamide labeled N-glycans with m/z 2443.994 and m/z 1295.013, respectively. While the N-glycan represented in Figure 4A corresponds to the G2S2 glycan: [5Hex + 4GlcNAc + 2NeuAc + +1

Procainamide + H] , the N-glycan represented in Figure 4B corresponds to the G2FS2 glycan: [5Hex + +2

4GlcNAc + 1Fuc + 2NeuAc + Procainamide + 2H] . No m/z 587.3 peak was observed in both MSMS spectra suggesting a lack of fucose residue (Figure 4A) and an antenna fucosylated N-glycan (Figure 4B). Rituximab N-glycan Fucosylation Assignment There is considerable global interest in biosimilar products in the biopharmaceutical industry. Hence, comprehensive analytical characterization is required to demonstrate that a biosimilar product is 9



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significantly comparable in quality, safety, efficacy and structure to the innovator drug. Rituximab is a chimeric IgG1 anti-CD20 monoclonal antibody (mAb) for treating B-cell non-Hodgkin’s lymphoma, rheumatoid arthritis and a wide range of immunologic illness. ®

47,48

®

Rituximab is available as Rituxan ,

®

Mabthera and Reditux in the U.S., Europe and India, respectively. Like many mAbs, Rituximab is glycosylated on a single N-glycosylation site. In this study, Rituximab was analyzed to demonstrate the application of the described technique to the characterization of therapeutic proteins. Figure 5A represents the fluorescent chromatogram of procainamide labeled Rituximab N-glycans while Figure 5B represents its corresponding tandem MS total ion chromatogram (TIC) data. The Rituximab N-glycan peak labels shown in Figure 5A are based on their observed masses and monosaccharide compositions as detailed in Table 3. All the fucosylated Rituximab N-glycans in Figure 5B are denoted by an asterisk (*) on the corresponding peaks. To verify the fucosylated type(s) present in the Rituximab N-glycan species, the XIC of m/z (587.3 ± 0.01) was constructed from Figure 5B. The m/z 587.3 XIC profile is represented in Figure 5C. As shown in Figure 5C, all the high abundant Rituximab fucosylated N-glycan peaks observed in Figure 5A and 5B have a corresponding m/z 587.3 peak in Figure 5C. It is also important to note that it was difficult to confirm the m/z 587.3 peaks in Figure 5C for the significantly low abundant N-glycan peaks of Rituximab. This particular result shows that the sensitivity of the proposed method needs to be further evaluated to determine the sensitivity threshold where low abundant N-glycan peaks can provide reliable tandem MS signals for their unambiguous identification as core- or antennafucosylated. MSMS Verification of Rituximab N-glycan Fucosylation Assignment The MSMS data of the Rituximab N-glycan peaks were further evaluated to confirm the glycan composition assignments made in Table 3 and to verify the fucose types present in the high abundant Nglycans of Rituximab. Figure 6 represents the MSMS spectra for a G0F Rituximab procainamide labeled +1

N-glycan with m/z 1682.714 corresponding in mass to [3Hex + 4GlcNAc + 1Fuc + Procainamide + H] . Particularly, the m/z 587.3 peak was observed in Figure 6 confirming that the investigated glycan is corefucosylated. Again, the m/z 790.4 peak (another core fucose diagnostic peak) was observed in Figure 6 further confirming core-fucosylation in the investigated N-glycan. 10


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Conclusion Procainamide labeling significantly increases fluorescent and MS sensitivities of N-glycans. Furthermore, tandem MS analysis of procainamide labeled N-glycans is shown here to provide a unique platform for the rapid assignment of fucosylation types in proteins. Procainamide labeling minimizes proton and fucose migration often associated with the tandem MS analysis of native and 2-AB labeled Nglycans. With fucose migration, the resulting tandem MS data of native or 2-AB labeled N-glycans leads to the random assignment of protein N-glycans as either core or antenna fucosylated. Reduction or elimination of fucose migration enables the assignment of protein fucosylation with less ambiguity. It is noteworthy that the described strategy involving procainamide labeling and tandem MS analysis towards the characterization of N-glycan fucosylation types is a simple technique. The extracted ion ®

chromatogram for m/z 587.3 from a sample’s tandem MS data (using the MassLynx algorithm) can be generated quickly, and reveals the unique fucosylation type(s) in the protein being investigated. This approach is a rapid means of assigning N-glycan fucosylation types without necessarily reviewing the individual tandem MS data of each N-glycan from the sample. The described technique is expected to find relevant applications in the characterization of therapeutic proteins, their biosimilars and in the search for disease biomarkers.

Acknowledgement Preliminary work on the development and optimization of procainamide labeling of N-glycans by Dr. Song Klapoetke (while at PPD) is gratefully acknowledged. Supporting Information Available: This material is available free of charge via the internet at http://pubs.acs.org.

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24. Niwa, R.; Natsume, A.; Uehara, A.; Wakitani, M.; Iida, S.; Uchida, K., J. Immunol. Methods 2005, 306, 151-160. 25. Niwa, R.; Sakurada, M.; Kobayashi, Y.; Uehara, A.; Matsushima, K.; Ueda, R., Clin. Cancer Res 2005, 11, 2327-2336. 26. Natsume, A.; Wakitani, M.; Yamane-Ohnuki, N.; Shoji-Hosaka, E.; Niwa, R.; Uchida, K., J. Immunol. Methods 2005, 306, 93-103. 27. Natsume, A.; Wakitani, M.; Yamane-Ohnuki, N.; Shoji-Hosaka, E.; Niwa, R.; Uchida, K., J. Biochem 2006, 140, 359-368. 28. Shoji-Hosaka, E.; Kobayashi, Y.; Wakitani, M.; Uchida, K.; Niwa, R.; Nakamura, K., J. Biochem 2006, 140, 777-783. 29. Masuda, K.; Kubota, T.; Kaneko, E.; Iida, S.; Wakitani, M.; Kobayashi-Natsume, Y., Mol. Immunol 2007, 44, 3122-3131. 30. Kanda, Y.; Yamane-Ohnuki, N.; Sakai, N.; Yamano, K.; Nakano, R.; Inoue, M., Biotechnol. Bioeng 2006, 94, 680-688. 31. Kanda, Y.; Imai-Nishiya, H.; Kuni-Kamochi, R.; Mori, K.; Inoue, M.; Kitajima-Miyama, K., J. Biotechnol 2007, 130, 300-310. 32. Kanda, Y.; Yamada, T.; Mori, K.; Okazaki, A.; Inoue, M.; Kitajima-Miyama, K., Glycobiology 2007, 17, 104-118. 33. Imai-Nishiya, H.; Mori, K.; Inoue, M.; Wakitani, M.; Iida, S.; Shitara, K., Biotechnol 2007, 7, 84. 34. Suzuki, E.; Niwa, R.; Saji, S.; Muta, M.; Hirose, M.; Iida, S., Clin. Cancer Res 2007, 13, 18751882. 35. Satoh, M.; Iida, S.; Shitara, K., Expert Opin. Biol. Ther 2006, 6, 1161-1173. 36. Iida, S.; Misaka, H.; Inoue, M.; Shibata, M.; Nakano, R.; Yamane-Ohnuki, N., Clin. Cancer Res 2006, 12, 2879-2887. 37. Wuhrer, M.; Koeleman, C. A.; Hokke, C. H.; Deelder, A. M., Rapid Commun Mass Spectrom 2006, 20 (11), 1747-1754. 38. Ernst, B.; Muller, D. R.; Richter, W. J., Intl. Journal of Mass Spectrom and Ion Processes 1997, 160 (1-3), 283-290. 39. Ailor, E.; Takahashi, N.; Tsukamoto, Y.; Masuda, K.; Rahman, B. A.; Jarvis, D. L., Lee, Y. C.; Betenbaugh, M. J., Glycobiology 2000, 10 (8), 837-847. 40. Haslam, S. M.; Coles, G. C.; Morris, H. R.; Dell, A., Glycobiology 2000, 10 (2), 223-229. 41. Klapoetke, S.; Zhang, J.; Becht, S.; Gu, X.; Ding, X., J. Pharm Biomed Anal 2011, 56, 513-520. 42. Aster, R. H., Blood 2104, 123, 463-464. 43. Nakano, M.; Kakehi, K.; Tsai, M.; Lee, Y. C., Glycobiology 2004, 14 (5), 431-441. 44. Klapoetke, S.; Zhang, J.; Becht, S.; Gu, X.; Ding, X., J. Pharm Biomed Anal 2010, 53, 315-324.

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Figure Caption Figure 1. Procainamide labeled human IgG N-glycan profiles (A) fluorescent chromatogram, (B) total ion chromatogram and (C) extracted ion chromatogram of the 587.3 Da core fucosylation diagnostic ion. Asterisks (*) in Figure 1B denote fucosylated Nglycans. +

Figure 2. MSMS spectra of procainamide labeled human IgG N-glycans: (A) G1F: (4Hex + 4GlcNAc + 1Fuc + Procainamide + H) + with core fucosylation and (B) G2: (5Hex + 4GlcNAc + Procainamide + H) with no fucosylation. Triangles ( ), rectangles ( ) and

circles ( and ) represent Fucose (Fuc), GlcNAc (N-Acetyl glucosamine) and Hexose (Hex) residues, respectively while the pink spheres ( ) represent the procainamide tag.

Figure 3. Procainamide labeled human alpha-1-acid glycoprotein N-glycan profiles (A) fluorescent chromatogram, (B) total ion chromatogram and (C) extracted ion chromatogram of the 587.3 Da core fucosylation diagnostic ion. Asterisks (*) in Figure 3B denote fucosylated N-glycans.

Figure 4. MSMS spectra of procainamide labeled human alpha-1-acid glycoprotein N-glycans: (A) G2S2 (5Hex + 4HexNAc + + + 2NeuAc + Procainamide + H) with no fucosylation. (B) G2FS2 (5Hex + 4HexNAc + 1Fuc + 2NeuAc + Procainamide + H) with antennae fucosylation. Triangles ( ), rectangles ( ) and circles ( and glucosamine) and Hexose (Hex) residues, respectively while the pink spheres (

) represent Fucose (Fuc), GlcNAc (N-Acetyl ) represent the procainamide tag.

Figure 5. Procainamide labeled Rituximab N-glycan profiles (A) fluorescent chromatogram, (B) total ion chromatogram and (C) extracted ion chromatogram of the 587.3 Da core fucosylation diagnostic ion. Asterisks (*) in Figure 5B denote fucosylated Nglycans. +

Figure 6. MSMS spectra of procainamide labeled G0F Rituximab N-glycan (3Hex + 4HexNAc + 1Fuc + Procainamide + H) with core fucosylation. Triangles ( ), rectangles ( ) and circles ( Hexose (Hex) residues, respectively while the pink spheres (

and

) represent Fucose (Fuc), GlcNAc (N-Acetyl glucosamine) and ) represent the procainamide tag.

Table Caption Table 1: List of procainamide labeled human IgG N-glycans Table 2: List of procainamide labeled human alpha-1-acid glycoprotein N-glycans Table 3: List of procainamide labeled Rituximab N-glycans

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Figure 1A, 1B and 1C

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Figure 2A and 2B

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Figure 4A and 4B

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Figure 6

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Table 1 Proposed Glycan ID G0F-N G0 G0F Man5 G1F-N G0F+N G1 G1F(a) G1F(b) G1F+N(a) G1F+N(b) G2 G2F G2F+N G1FS1 G2S1 G2FS1

Fluorescent Retention Time (min) 19.582 20.463 22.449 23.644 23.644 24.290 24.876 26.160 26.698 27.503 27.997 28.487 30.104 30.965 31.687 33.208 34.607

Mass/Charge (m/z)

Charge State

1479.637 1536.661 1682.713 1454.627 1641.712 1885.805 1698.721 1844.775 1844.762 2047.868 2047.858 1860.761 2006.833 2209.922 2135.884 2151.881 2297.925

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Mass/Charge (m/z)

Charge State

1860.781 1787.036 2151.873 2151.873 1932.783 2151.852 2443.001 1295.013 1404.552 1404.555 1477.596 1550.090 1550.093

1 1 1 1 1 1 1 2 2 2 2 2 2

Mass/Charge (m/z)

Charge State

1479.661 1536.676 1682.690 1454.599 1641.689 1844.647 1844.645 2006.837 1149.464 1149.464 1294.999

1 1 1 1 1 1 1 1 2 2 2

Glycan Composition Hexose 3 3 3 5 4 3 4 4 4 4 4 5 5 5 4 5 5

GlcNAc 3 4 4 2 3 5 4 4 4 5 5 4 4 5 4 4 4

Hexose 5 4 5 5 4 5 5 5 6 6 6 6 6

GlcNAc 4 3 4 4 3 4 4 4 5 5 5 5 5

Fucose 1 0 1 0 1 1 0 1 1 1 1 0 1 1 1 0 1

NeuAc 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1

Table 2 Proposed Glycan ID G2 G1S1-N G2S1(a) G2S1(b) G1FS1-N G2S1 G2S2 G2FS2 G3S2(a) G3S2(b) G3FS2 G3S3(a) G3S3(b)

Fluorescent Retention Time (min) 28.481 31.386 33.024 33.200 34.576 36.005 37.484 38.703 39.823 41.094 41.531 43.356 44.838

Glycan Composition Fucose 0 0 0 0 1 0 0 1 0 0 1 0 0

NeuAc 0 1 1 1 1 1 2 2 2 2 2 3 3

Table 3 Proposed Glycan ID G0F-N G0 G0F Man5 G1F-N G1F(a) G1F(b) G2F G2FS1(a) G2FS1(b) G2FS2

Fluorescent Retention Time (min) 19.589 20.472 22.452 23.581 24.216 26.156 26.695 30.094 32.723 33.058 35.714

Glycan Composition Hexose 3 3 3 5 4 4 4 5 5 5 5

GlcNAc 3 4 4 2 3 4 4 4 4 4 4

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Fucose 1 0 1 0 1 1 1 1 1 1 1

NeuAc 0 0 0 0 0 0 0 0 1 1 2

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Table of Content

23


Assignment of Core versus Antenna Fucosylation Types in Protein N

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