- Email: [email protected]
Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans
Accepted Manuscript Comparison of Fluorescent Tags for Analysis of Mannose-6-phosphate Glycans Ji-Yeon Kang, Ohsuk Kwon, Jin Young Gil, Doo-Byoung Oh PII:
S0003-2697(16)00056-7
DOI:
10.1016/j.ab.2016.02.004
Reference:
YABIO 12310
To appear in:
Analytical Biochemistry
Received Date: 12 September 2015 Revised Date:
3 February 2016
Accepted Date: 3 February 2016
Please cite this article as: J.-Y. Kang, O. Kwon, J.Y. Gil, D.-B. Oh, Comparison of Fluorescent Tags for Analysis of Mannose-6-phosphate Glycans, Analytical Biochemistry (2016), doi: 10.1016/ j.ab.2016.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison of Fluorescent Tags for Analysis of Mannose-6phosphate Glycans
1
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of
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Bioscience and Biotechnology (KRIBB), Daejeon, Korea. 2
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Ji-Yeon Kang1, Ohsuk Kwon1,2, Jin Young Gil1, and Doo-Byoung Oh1,2,*
Biosystems and Bioengineering Program, University of Science and Technology (UST),
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Daejeon, Korea.
Running Title: Mannose-6-phosphate Glycan Analysis
*
Doo-Byoung Oh
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Corresponding author
Korea Research Institute of Bioscience and Biotechnology (KRIBB)
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125 Gwahakro, Yuseong-gu, Daejeon 34141, Korea
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Tel: 82-42-860-4457, Fax: 82-42-879-8494, E-mail: [email protected]
1
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Abstract Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans
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containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-
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amino)-6-aminoacridine [AA-Ac]) were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.
Keywords: Mannose-6-phosphate, Glycan analysis, Fluorescent labeling tag, High
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performance liquid chromatography (HPLC), Matrix-assisted laser desorption/ionization
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time-of-flight (MALDI-TOF) mass spectrometry
Abbreviations used: 2-AA, 2-aminobenzoic acid; AA-Ac, 3-(acetyl-amino)-6-aminoacridine;
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2-AB, 2-aminobenzamide; AMAC, 2-aminoacridone; CHO, Chinese hamster ovary; HILIC, hydrophilic
interaction
chromatography;
M-6-P,
liquid
chromatography;
mannose-6-phosphate;
HPLC,
MALDI-TOF,
desorption/ionization time-of-flight; MAH (mild acid hydrolysis)
2
high-performance matrix-assisted
liquid laser
ACCEPTED MANUSCRIPT Many therapeutic proteins are glycoproteins and the attached glycans play important roles in therapeutic efficacy, in vivo half-lives, tissue distribution, and immune reaction. Glycan analysis is important in the characterization and quality control of glycoproteins for
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therapeutic uses [1] and various analytical methods have been developed. High-performance anion-exchange chromatography coupled with pulsed amperometric detection was widely used for glycan analyses without derivatization, but it suffers from an unstable baseline, low
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reproducibility, and use of high pH and salt. Instead, high-performance liquid chromatography (HPLC) analysis of fluorescently labeled glycans has become popular
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because of the generation of robust and reproducible results together with highly sensitive detection [2]. Most fluorescent tags used are aromatic amines labeling glycans at the reducing end by reductive amination via a Schiff base. Although 2-aminopyridine was widely used because of a historical reason (the first introduction in 1978), it seems to have disadvantages
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including relatively low sensitivity and a cumbersome labeling and purification process [2]. Currently, 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA) are the most popular tags in glycan analysis. Although other tags such as 3-(acetyl-amino)-6-aminoacridine (AA-
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Ac) and 2-aminoacridone (AMAC) were introduced as especially sensitive tags [3], many of them including AMAC were reported to be less sensitive than 2-AA in another study [4].
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Lysosomal storage diseases are caused by genetic defects that are accompanied by lysosomal hydrolases deficiencies, which lead to massive accumulation of undigested compounds. Among more than 40 lysosomal storage diseases, seven diseases (Gaucher, Fabry, and Pompe diseases and mucopolysaccharidosis type I, II, IVA, and VI) are successfully treated with enzyme replacement therapy using the recombinant lysosomal enzyme [5]. These enzymes (except the ones for Gaucher disease) require glycans containing mannose-6phosphates (M-6-Ps), which are recognized by the M-6-P receptor on the plasma membrane 3
ACCEPTED MANUSCRIPT for lysosomal targeting. Analysis of M-6-P glycans is important because the structure and content of the M-6-P glycans determine the efficiency of lysosomal targeting and, therefore, therapeutic efficacy [6; 7]. However, only a few analytical methods for M-6-P glycans have
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been reported until now [8; 9; 10; 11]. Monosaccharide analysis was used for measurement of M-6-P quantity, but it cannot provide information about the structures of the M-6-P glycans [10; 11]. Reverse phase column HPLC with mass spectrometry has been used for the analyses
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of glycopeptide containing M-6-P glycans: notably, the glycopeptides harboring mono- or biphosphorylated glycans were well separated and identified [11; 12]. However, these studies
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focused on analysis of glycopeptide rather than on analysis of the M-6-P glycan structure. HPLC profiles of 2-AA-labeled glycans of lysosomal acid lipases were obtained using an amine column [9]. The Dr. Rudd group applied ultra-performance liquid chromatography equipped with a hydrophilic interaction liquid chromatography (HILIC) column for analysis
cells [8].
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of 2-AB-labeled glycans of β-glucuronidase produced from Chinese hamster ovary (CHO)
Most of the approved therapeutic enzymes have been produced from mammalian
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cells including CHO cells and human fibroblasts, which have a pathway to synthesize M-6-P glycans. Although yeasts do not have glycans containing M-6-Ps, some of their glycans are
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attached to mannnoyslphosphate residue, which forms mannosylphosphorylated mannose (mannose-1-phosphate-6-O-mannnose). Through an uncapping process to remove the outer capping mannose residue, this structure can be converted to M-6-P (phosphate-6-Omannnose). Several groups successfully engineered yeasts to produce enzymes attached with M-6-P glycans [6; 13; 14; 15]. The conversions from the mannosylphosphorylated structures to M-6-P glycans have been analyzed by HPLC and capillary electrophoresis methods after the fluorescent-labeling of the glycans [6; 13; 14; 15]. 4
ACCEPTED MANUSCRIPT Recently, our group constructed a glyco-engineered Saccharomyces cerevisiae mnn1∆och1∆ strain overexpressing YlMPO1 gene (Scmnn1∆och1∆/YlMPO1) that was shown to have very high content of mannosylphosphorylated glycans [15]. To analyze the
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conversion from these glycans to M-6-P glycans, we used a DNA sequencer-based method and amine column HPLC with 2-AA labeling of glycans. However, in HPLC analysis, we were hardly able to detect bi-phosphorylated glycan (containing two M-6-P structures), which
ability and efficacy of therapeutic enzymes [6; 16].
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is a high affinity structure for the M-6-P receptor and determines the lysosomal targeting
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In this study, we optimized HPLC conditions for the analysis of M-6-P glycans (especially containing two M-6-Ps) with 2-AA labeling. Mannosylphosphorylated N-glycans were prepared from yeast cell wall mannoproteins and labeled with 2-AA (Sigma-Aldrich, St. Louis, MO, USA) as described previously [15]. The 2-AA-labeled glycans were analyzed by
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a Shodex Asahipak NH2P-50 amine column (5 µm, 4.6 mm x 250 mm) purchased from Showa Denko (Tokyo, Japan) using a Waters Alliance system equipped with a Waters 2475 fluorescence detector (Milford, MA, USA). Initially, the column was equilibrated with 90%
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solvent A (acetonitrile containing 2% acetic acid and 1% tetrahydrofuran) and 10% solvent B (5% acetic acid, 3% triethylamine, and 1% tetrahydrofuran in water), which are generally
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used solvents for 2-AA-labeled glycan analysis. After injecting a 10 µl sample, elution was carried out with a linear gradient from 10% solvent B (90% solvent A) to 90% solvent B (10% solvent A) at a flow rate of 1 ml/min for 70 min at 50°C. Fluorescence of 2-AA was monitored with 360 nm excitation and 425 nm emission. The eluted peak fractions were collected and their masses were identified by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry as previously described [15; 17; 18]. The amine column separated neutral (Man8GlcNAc2), mono- (Man-P-Man8GlcNAc2), and bi5
ACCEPTED MANUSCRIPT mannosylphosphorylated [(Man-P)2-Man8GlcNAc2] glycans in accordance with their charge and size (upper panel in Fig. 1A). The outer mannose residue of the mannosylphosphorylated glycans was uncapped by
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a mild acid hydrolysis (MAH) to generate M-6-P glycans (lower panel in Fig. 1A). This uncapping process converted mono-mannosylphosphorylated glycan to mono-phosphorylated glycan containing one M-6-P structure (P-Man8GlcNAc2) as expected. However, bi-
even with a prolonged elution time (up to 150 min).
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phosphorylated glycan containing two M-6-P structures (P2-Man8GlcNAc2) was not detected,
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We assumed that the bi-phosphorylated glycan was not eluted from the Shodex Asahipak NH2P-50 amine column because of increased negative charges of the phosphate group resulting from removal of the outer mannose residues. In order to elute the biphosphorylated glycan from the column, we enhanced the hydrophilicity of solvent B by
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increasing the concentrations of acetic acid and triethlyamine (solvent B-h; 10% acetic acid, 6% triethylamine, and 1% tetrahydrofuran in water). When the elution was carried out with a gradient from 10% solvent B-h to 90% solvent B-h, the bi-phosphorylated glycan peak was
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detected (lower panel in Fig. 1B). The identities of all peaks were confirmed by measuring the masses of the collected peak eluates (see Fig. 2 in Ref [19]). In contrast with our work,
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Du et al. reported that the bi-phosphorylated glycan was detected in amine column HPLC using the generally used solvents [9]. They analyzed 2-AA-labeled glycans prepared from lysosomal acid lipase using a Luna NH2 amine column, while we used a Shodex Asahipak NH2P-50 amine column. In their work, the bi-phosphorylated glycan was detected as a broad peak at ~100 min, which is far later than the mono-phosphorylated peaks at around 52-54 min. In contrast, the bi-phosphorylated glycan was detected at 55 min in our analysis, which is only less than 10 min behind the mono-phosphorylated peaks (lower panel in Fig. 1B). Our 6
ACCEPTED MANUSCRIPT optimized analysis condition using the more hydrophilic solvent B-h enabled efficient detection of the bi-phosphorylated glycan with 2-AA labeling. We investigated whether the MAH condition (incubation in 0.5 M formic acid at
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80°C for 1 hour) affects the content of bi-phosphorylated glycan. When compared with MAH condition using hydrochloric acid (incubation in 0.01 M HCl at 100°C for 1 hour) described in the previous works [16; 20], our MAH condition using formic acid enabled more sensitive
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detection and showed higher relative content of bi-phosphorylated glycan (Fig. 1E). Further, we checked the possibility that MAH may convert bi-phosphorylated glycan to mono-
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phosphorylated one. It was achieved by repeating MAH and then measuring the content of biphosphorylated glycans (Fig. 1F). Repeated MAH made no significant change in the content of bi-phosphorylated glycans, which suggests that the bi-phosphorylated glycan was not converted to other structures under our MAH condition.
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After optimization of the solvent in HPLC analysis for bi-phosphorylated glycan with 2-AA labeling, we conducted further investigations to determine whether other fluorescent tags would also benefit from adjustment of the solvents. The same glycans were
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labeled with the frequently used fluorescent tags 2-AB and AA-Ac and then analyzed using solvents A and B (see Fig. 3 in Ref [19]). In contrast with the results for 2-AA-labeled
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glycans, the bi-phosphorylated glycan was detected in the HPLC profiles of both 2-AB- and AA-Ac-labeled glycans. Notably, AA-Ac-labeled glycan peaks were eluted earlier than 2AB-labeled glycan peaks (compare Figs. 3A and B in Ref [19]). Taken together, these results suggest that the hydrophilicity of fluorescent tags affects the elution time in HPLC together with the hydrophilicity of the elution solvent. AA-Ac-labeled glycans are eluted earlier from the amine column because AA-Ac, as a result of its possession of three aromatic rings, has more hydrophobic features than 2-AB and 2-AA with one aromatic ring each (see Fig. 1 in 7
ACCEPTED MANUSCRIPT Ref [19]). The need for the more hydrophilic elution solvent B-h in 2-AA-labeled glycan analysis for detection of bi-phosphorylated glycan can be explained by the additional negative charge of the carboxyl group of the 2-AA tag.
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We conducted further comparative investigations to determine any differences among these three fluorescent tags for detecting and quantifying the bi-phosphorylated glycan. For direct comparison, all of the HPLC profiles were analyzed using the same
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solvents (A and B-h) optimized for 2-AA-labeled glycan analysis (Fig. 1C), which resulted in reduced elution times for 2-AB- and AA-Ac-labeled M-6-P glycans (compare Fig. 1C in this
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article and Fig. 3A in Ref [19]). Especially, the bi-phosphorylated glycan labeled with 2-AB was eluted at 52 min with the use of solvents A and B-h, which is a substantial decrease in the elution time (6 min) compared with the time of 58 min in the analysis using solvents A and B. From the integrated peak areas, the relative contents of neutral and phosphorylated
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glcyans were calculated (Fig. 1D). The bi-phosphorylated glycan has the highest relative content value in the 2-AA-labeling analysis (41%), and this value is much higher than the values from the 2-AB- (32%) and AA-Ac-labeling analyses (32%). The relative content value
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of bi-phosphorylated glycan in the 2-AA-labeling analysis is more comparable to the result calculated from DNA sequencer-based analysis [15], while the 2-AB- and AA-Ac labeling
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appear to underestimate the relative content of bi-phosphorylated glycan. Taken together, our results show that the elution time and quantification of the bi-phosphorylated glycan peak in HPLC analysis are affected greatly by both hydrophilicities of the fluorescent tag for labeling and the elution solvent used in HPLC analysis, which suggests that careful consideration is required in analyzing M-6-P glycans of therapeutic enzymes. M-6-P glycans converted from mannosylphosphorylated glycans were also analyzed by MALDI-TOF mass spectrometry (see Fig. 4 in Ref [19]). Notably, native M-6-P glycans 8
ACCEPTED MANUSCRIPT (without derivatization) were not detected in our hands (data not shown) because the negative charge of M-6-P requires the use of a linear negative ion mode, which is insensitive compared with a positive ion mode. In contrast, all of 2-AA, 2-AB, and AA-Ac labeling
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increased the sensitivity and enabled the detection of M-6-P glycans (Fig. 2A). We found that the enhanced detection sensitivities of the bi-phosphorylated glycan differed depending on the fluorescent tag. The intensity of bi-phosphorylated glycan was normalized with the
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intensity of mono-phosphorylated glycan ([Intensity of bi-phsphorylated glycan]/[Intensity of mono-phosphorylated glycan]) and the highest value (1.6) was shown by 2-AA labeling,
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indicating the highest amount of bi-phosphorylated glycan (Fig. 2B). This is in good agreement with the HPLC result using 2-AA labeling as well as with the previous DNA sequencer-based analysis result [15]. In contrast, as a result of insensitive detection of biphosphorylated glycan, the normalized intensity values for the bi-phosphorylated glycan in
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the analyses with 2-AB and AA-Ac labeling were less than 1.0 at only 0.81 and 0.90, which would lead to an underestimation of bi-phosphoorylated glycan. Moreover, only 2-AA labeling allows simultaneous analysis of neutral and phosphorylated glycans because neutral
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glycans can be detected in a negative ion mode by the negative charge of 2-AA (Fig. 2A). These results clearly indicate the superior performance of 2-AA labeling for M-6-P glycan
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analysis using MALDI-TOF mass spectrometry. In this study, we showed that the optimization of elution solvent hydrophilicity is
necessary for HPLC analysis of M-6-P glycans with 2-AA labeling. After the optimization, 2AA labeling was superior for quantifying the content of bi-phosphorylated glycan compared with 2-AB and AA-AC labeling. Moreover, 2-AA labeling showed the highest sensitivity in detection of the bi-phosphorylated glycan in MALDI-TOF MS analysis.
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Acknowledgement This work was supported by the Next-Generation BioGreen 21 Program (PJ011078) of the
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Rural Development Administration in Korea, and the National Research Foundation of Korea
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[NRF-2013M3A9B6075888].
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[13] H. Akeboshi, Y. Kasahara, D. Tsuji, K. Itoh, H. Sakuraba, Y. Chiba, and Y. Jigami, Production of human beta-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene. Glycobiology 19 (2009) 1002-9. [14] Y. Chiba, H. Sakuraba, M. Kotani, R. Kase, K. Kobayashi, M. Takeuchi, S. Ogasawara,
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Y. Maruyama, T. Nakajima, Y. Takaoka, and Y. Jigami, Production in yeast of alphagalactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease. Glycobiology 12 (2002) 821-8.
[15] J.Y. Gil, J.N. Park, K.J. Lee, J.Y. Kang, Y.H. Kim, S. Kim, S.Y. Kim, O. Kwon, Y.T. Lim,
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H.A. Kang, and D.B. Oh, Increased Mannosylphosphorylation of N-glycans by Heterologous Expression of YlMPO1 in Glyco-engineered Saccharomyces cerevisiae for Mannose-6-
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phosphate Modification. J Biotechnol (2015). [16] X. Song, Y. Lasanajak, L.J. Olson, M. Boonen, N.M. Dahms, S. Kornfeld, R.D. Cummings, and D.F. Smith, Glycan microarray analysis of P-type lectins reveals distinct phosphomannose glycan recognition. J Biol Chem 284 (2009) 35201-14. [17] K.J. Lee, J.Y. Gil, S.Y. Kim, O. Kwon, K. Ko, D.I. Kim, D.K. Kim, H.H. Kim, and D.B. Oh, Molecular characterization of acidic peptide:N-glycanase from the dimorphic yeast Yarrowia lipolytica. J Biochem 157 (2015) 35-43. [18] J.Y. Mun, K.J. Lee, H. Seo, M.S. Sung, Y.S. Cho, S.G. Lee, O. Kwon, and D.B. Oh, 12
ACCEPTED MANUSCRIPT Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis. Anal Chem 85 (2013) 7462-70. [19] J.Y. Kang, O. Kwon, J.Y. Gil, and D.B. Oh, Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags. Data in Brief (2016) submitted. [20] G.A. Baumbach, P.T. Saunders, F.W. Bazer, and R.M. Roberts, Uteroferrin has N-
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asparagine-linked high-mannose-type oligosaccharides that contain mannose 6-phosphate.
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Figure legends Fig. 1. HPLC analysis of the 2-AA-labeled M-6-P glycans. The 2-AA-labeled glycans obtained from Scmnn1∆och1∆/YlMPO1 were analyzed by amine column HPLC using either
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the generally used solvents (A) or the optimized solvents (B). After uncapping (lower panels), the glycan containing two M-6-Ps (indicated by the arrow) was only detected in the HPLC profile with the optimized solvent system (B). (C) 2-AA-, 2-AB-, and AA-Ac-labeled M-6-P
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glycans were analyzed using the same solvents optimized for 2-AA-labeling. (D) Relative contents (%) of neutral (Man7-9GlcNAc2, white bar), mono-phosphorylated (P-Man8GlcNAc2,
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gray bar), and bi-phosphorylated (P2-Man8GlcNAc2, black bar) glycans were calculated from the integrated peak areas. (E) For comparison of MAH conditions, the relative contents of biphosphorylated glycan (P2-Man8GlcNAc2) were obtained from MAH conditions using formic acid (gray bar) or hydrochloric acid (black bar). (F) The relative contents of bi-
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phosphorylated glycans were obtained from single (gray bar) or repeated MAHs (white bar). The bars represent the average of three replicated experiments. Asterisks indicate statistically significant differences (p < 0.05). Symbols used for glycans are those suggested
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by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). Green circle: mannose, blue square: GlcNAc, P: phosphate. Unidentified peaks are represented by *.
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(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. MALDI-TOF mass spectrometry analysis of M-6-P glycans. (A) Mass spectra of 2AA-, 2-AB-, and AA-Ac-labeled glycans were obtained. (B) Relative intensity ratios were calculated from [P2-Man8GlcNAc2 intensity]/ [P-Man8GlcNAc2 intensity]. Symbols are identical to those used in Fig. 1. 14
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S0003-2697(16)00056-7
DOI:
10.1016/j.ab.2016.02.004
Reference:
YABIO 12310
To appear in:
Analytical Biochemistry
Received Date: 12 September 2015 Revised Date:
3 February 2016
Accepted Date: 3 February 2016
Please cite this article as: J.-Y. Kang, O. Kwon, J.Y. Gil, D.-B. Oh, Comparison of Fluorescent Tags for Analysis of Mannose-6-phosphate Glycans, Analytical Biochemistry (2016), doi: 10.1016/ j.ab.2016.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison of Fluorescent Tags for Analysis of Mannose-6phosphate Glycans
1
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of
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Bioscience and Biotechnology (KRIBB), Daejeon, Korea. 2
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Ji-Yeon Kang1, Ohsuk Kwon1,2, Jin Young Gil1, and Doo-Byoung Oh1,2,*
Biosystems and Bioengineering Program, University of Science and Technology (UST),
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Daejeon, Korea.
Running Title: Mannose-6-phosphate Glycan Analysis
*
Doo-Byoung Oh
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Corresponding author
Korea Research Institute of Bioscience and Biotechnology (KRIBB)
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125 Gwahakro, Yuseong-gu, Daejeon 34141, Korea
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Tel: 82-42-860-4457, Fax: 82-42-879-8494, E-mail: [email protected]
1
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Abstract Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans
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containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-
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amino)-6-aminoacridine [AA-Ac]) were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.
Keywords: Mannose-6-phosphate, Glycan analysis, Fluorescent labeling tag, High
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performance liquid chromatography (HPLC), Matrix-assisted laser desorption/ionization
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time-of-flight (MALDI-TOF) mass spectrometry
Abbreviations used: 2-AA, 2-aminobenzoic acid; AA-Ac, 3-(acetyl-amino)-6-aminoacridine;
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2-AB, 2-aminobenzamide; AMAC, 2-aminoacridone; CHO, Chinese hamster ovary; HILIC, hydrophilic
interaction
chromatography;
M-6-P,
liquid
chromatography;
mannose-6-phosphate;
HPLC,
MALDI-TOF,
desorption/ionization time-of-flight; MAH (mild acid hydrolysis)
2
high-performance matrix-assisted
liquid laser
ACCEPTED MANUSCRIPT Many therapeutic proteins are glycoproteins and the attached glycans play important roles in therapeutic efficacy, in vivo half-lives, tissue distribution, and immune reaction. Glycan analysis is important in the characterization and quality control of glycoproteins for
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therapeutic uses [1] and various analytical methods have been developed. High-performance anion-exchange chromatography coupled with pulsed amperometric detection was widely used for glycan analyses without derivatization, but it suffers from an unstable baseline, low
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reproducibility, and use of high pH and salt. Instead, high-performance liquid chromatography (HPLC) analysis of fluorescently labeled glycans has become popular
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because of the generation of robust and reproducible results together with highly sensitive detection [2]. Most fluorescent tags used are aromatic amines labeling glycans at the reducing end by reductive amination via a Schiff base. Although 2-aminopyridine was widely used because of a historical reason (the first introduction in 1978), it seems to have disadvantages
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including relatively low sensitivity and a cumbersome labeling and purification process [2]. Currently, 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA) are the most popular tags in glycan analysis. Although other tags such as 3-(acetyl-amino)-6-aminoacridine (AA-
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Ac) and 2-aminoacridone (AMAC) were introduced as especially sensitive tags [3], many of them including AMAC were reported to be less sensitive than 2-AA in another study [4].
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Lysosomal storage diseases are caused by genetic defects that are accompanied by lysosomal hydrolases deficiencies, which lead to massive accumulation of undigested compounds. Among more than 40 lysosomal storage diseases, seven diseases (Gaucher, Fabry, and Pompe diseases and mucopolysaccharidosis type I, II, IVA, and VI) are successfully treated with enzyme replacement therapy using the recombinant lysosomal enzyme [5]. These enzymes (except the ones for Gaucher disease) require glycans containing mannose-6phosphates (M-6-Ps), which are recognized by the M-6-P receptor on the plasma membrane 3
ACCEPTED MANUSCRIPT for lysosomal targeting. Analysis of M-6-P glycans is important because the structure and content of the M-6-P glycans determine the efficiency of lysosomal targeting and, therefore, therapeutic efficacy [6; 7]. However, only a few analytical methods for M-6-P glycans have
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been reported until now [8; 9; 10; 11]. Monosaccharide analysis was used for measurement of M-6-P quantity, but it cannot provide information about the structures of the M-6-P glycans [10; 11]. Reverse phase column HPLC with mass spectrometry has been used for the analyses
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of glycopeptide containing M-6-P glycans: notably, the glycopeptides harboring mono- or biphosphorylated glycans were well separated and identified [11; 12]. However, these studies
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focused on analysis of glycopeptide rather than on analysis of the M-6-P glycan structure. HPLC profiles of 2-AA-labeled glycans of lysosomal acid lipases were obtained using an amine column [9]. The Dr. Rudd group applied ultra-performance liquid chromatography equipped with a hydrophilic interaction liquid chromatography (HILIC) column for analysis
cells [8].
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of 2-AB-labeled glycans of β-glucuronidase produced from Chinese hamster ovary (CHO)
Most of the approved therapeutic enzymes have been produced from mammalian
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cells including CHO cells and human fibroblasts, which have a pathway to synthesize M-6-P glycans. Although yeasts do not have glycans containing M-6-Ps, some of their glycans are
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attached to mannnoyslphosphate residue, which forms mannosylphosphorylated mannose (mannose-1-phosphate-6-O-mannnose). Through an uncapping process to remove the outer capping mannose residue, this structure can be converted to M-6-P (phosphate-6-Omannnose). Several groups successfully engineered yeasts to produce enzymes attached with M-6-P glycans [6; 13; 14; 15]. The conversions from the mannosylphosphorylated structures to M-6-P glycans have been analyzed by HPLC and capillary electrophoresis methods after the fluorescent-labeling of the glycans [6; 13; 14; 15]. 4
ACCEPTED MANUSCRIPT Recently, our group constructed a glyco-engineered Saccharomyces cerevisiae mnn1∆och1∆ strain overexpressing YlMPO1 gene (Scmnn1∆och1∆/YlMPO1) that was shown to have very high content of mannosylphosphorylated glycans [15]. To analyze the
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conversion from these glycans to M-6-P glycans, we used a DNA sequencer-based method and amine column HPLC with 2-AA labeling of glycans. However, in HPLC analysis, we were hardly able to detect bi-phosphorylated glycan (containing two M-6-P structures), which
ability and efficacy of therapeutic enzymes [6; 16].
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is a high affinity structure for the M-6-P receptor and determines the lysosomal targeting
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In this study, we optimized HPLC conditions for the analysis of M-6-P glycans (especially containing two M-6-Ps) with 2-AA labeling. Mannosylphosphorylated N-glycans were prepared from yeast cell wall mannoproteins and labeled with 2-AA (Sigma-Aldrich, St. Louis, MO, USA) as described previously [15]. The 2-AA-labeled glycans were analyzed by
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a Shodex Asahipak NH2P-50 amine column (5 µm, 4.6 mm x 250 mm) purchased from Showa Denko (Tokyo, Japan) using a Waters Alliance system equipped with a Waters 2475 fluorescence detector (Milford, MA, USA). Initially, the column was equilibrated with 90%
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solvent A (acetonitrile containing 2% acetic acid and 1% tetrahydrofuran) and 10% solvent B (5% acetic acid, 3% triethylamine, and 1% tetrahydrofuran in water), which are generally
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used solvents for 2-AA-labeled glycan analysis. After injecting a 10 µl sample, elution was carried out with a linear gradient from 10% solvent B (90% solvent A) to 90% solvent B (10% solvent A) at a flow rate of 1 ml/min for 70 min at 50°C. Fluorescence of 2-AA was monitored with 360 nm excitation and 425 nm emission. The eluted peak fractions were collected and their masses were identified by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry as previously described [15; 17; 18]. The amine column separated neutral (Man8GlcNAc2), mono- (Man-P-Man8GlcNAc2), and bi5
ACCEPTED MANUSCRIPT mannosylphosphorylated [(Man-P)2-Man8GlcNAc2] glycans in accordance with their charge and size (upper panel in Fig. 1A). The outer mannose residue of the mannosylphosphorylated glycans was uncapped by
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a mild acid hydrolysis (MAH) to generate M-6-P glycans (lower panel in Fig. 1A). This uncapping process converted mono-mannosylphosphorylated glycan to mono-phosphorylated glycan containing one M-6-P structure (P-Man8GlcNAc2) as expected. However, bi-
even with a prolonged elution time (up to 150 min).
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phosphorylated glycan containing two M-6-P structures (P2-Man8GlcNAc2) was not detected,
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We assumed that the bi-phosphorylated glycan was not eluted from the Shodex Asahipak NH2P-50 amine column because of increased negative charges of the phosphate group resulting from removal of the outer mannose residues. In order to elute the biphosphorylated glycan from the column, we enhanced the hydrophilicity of solvent B by
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increasing the concentrations of acetic acid and triethlyamine (solvent B-h; 10% acetic acid, 6% triethylamine, and 1% tetrahydrofuran in water). When the elution was carried out with a gradient from 10% solvent B-h to 90% solvent B-h, the bi-phosphorylated glycan peak was
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detected (lower panel in Fig. 1B). The identities of all peaks were confirmed by measuring the masses of the collected peak eluates (see Fig. 2 in Ref [19]). In contrast with our work,
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Du et al. reported that the bi-phosphorylated glycan was detected in amine column HPLC using the generally used solvents [9]. They analyzed 2-AA-labeled glycans prepared from lysosomal acid lipase using a Luna NH2 amine column, while we used a Shodex Asahipak NH2P-50 amine column. In their work, the bi-phosphorylated glycan was detected as a broad peak at ~100 min, which is far later than the mono-phosphorylated peaks at around 52-54 min. In contrast, the bi-phosphorylated glycan was detected at 55 min in our analysis, which is only less than 10 min behind the mono-phosphorylated peaks (lower panel in Fig. 1B). Our 6
ACCEPTED MANUSCRIPT optimized analysis condition using the more hydrophilic solvent B-h enabled efficient detection of the bi-phosphorylated glycan with 2-AA labeling. We investigated whether the MAH condition (incubation in 0.5 M formic acid at
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80°C for 1 hour) affects the content of bi-phosphorylated glycan. When compared with MAH condition using hydrochloric acid (incubation in 0.01 M HCl at 100°C for 1 hour) described in the previous works [16; 20], our MAH condition using formic acid enabled more sensitive
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detection and showed higher relative content of bi-phosphorylated glycan (Fig. 1E). Further, we checked the possibility that MAH may convert bi-phosphorylated glycan to mono-
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phosphorylated one. It was achieved by repeating MAH and then measuring the content of biphosphorylated glycans (Fig. 1F). Repeated MAH made no significant change in the content of bi-phosphorylated glycans, which suggests that the bi-phosphorylated glycan was not converted to other structures under our MAH condition.
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After optimization of the solvent in HPLC analysis for bi-phosphorylated glycan with 2-AA labeling, we conducted further investigations to determine whether other fluorescent tags would also benefit from adjustment of the solvents. The same glycans were
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labeled with the frequently used fluorescent tags 2-AB and AA-Ac and then analyzed using solvents A and B (see Fig. 3 in Ref [19]). In contrast with the results for 2-AA-labeled
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glycans, the bi-phosphorylated glycan was detected in the HPLC profiles of both 2-AB- and AA-Ac-labeled glycans. Notably, AA-Ac-labeled glycan peaks were eluted earlier than 2AB-labeled glycan peaks (compare Figs. 3A and B in Ref [19]). Taken together, these results suggest that the hydrophilicity of fluorescent tags affects the elution time in HPLC together with the hydrophilicity of the elution solvent. AA-Ac-labeled glycans are eluted earlier from the amine column because AA-Ac, as a result of its possession of three aromatic rings, has more hydrophobic features than 2-AB and 2-AA with one aromatic ring each (see Fig. 1 in 7
ACCEPTED MANUSCRIPT Ref [19]). The need for the more hydrophilic elution solvent B-h in 2-AA-labeled glycan analysis for detection of bi-phosphorylated glycan can be explained by the additional negative charge of the carboxyl group of the 2-AA tag.
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We conducted further comparative investigations to determine any differences among these three fluorescent tags for detecting and quantifying the bi-phosphorylated glycan. For direct comparison, all of the HPLC profiles were analyzed using the same
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solvents (A and B-h) optimized for 2-AA-labeled glycan analysis (Fig. 1C), which resulted in reduced elution times for 2-AB- and AA-Ac-labeled M-6-P glycans (compare Fig. 1C in this
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article and Fig. 3A in Ref [19]). Especially, the bi-phosphorylated glycan labeled with 2-AB was eluted at 52 min with the use of solvents A and B-h, which is a substantial decrease in the elution time (6 min) compared with the time of 58 min in the analysis using solvents A and B. From the integrated peak areas, the relative contents of neutral and phosphorylated
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glcyans were calculated (Fig. 1D). The bi-phosphorylated glycan has the highest relative content value in the 2-AA-labeling analysis (41%), and this value is much higher than the values from the 2-AB- (32%) and AA-Ac-labeling analyses (32%). The relative content value
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of bi-phosphorylated glycan in the 2-AA-labeling analysis is more comparable to the result calculated from DNA sequencer-based analysis [15], while the 2-AB- and AA-Ac labeling
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appear to underestimate the relative content of bi-phosphorylated glycan. Taken together, our results show that the elution time and quantification of the bi-phosphorylated glycan peak in HPLC analysis are affected greatly by both hydrophilicities of the fluorescent tag for labeling and the elution solvent used in HPLC analysis, which suggests that careful consideration is required in analyzing M-6-P glycans of therapeutic enzymes. M-6-P glycans converted from mannosylphosphorylated glycans were also analyzed by MALDI-TOF mass spectrometry (see Fig. 4 in Ref [19]). Notably, native M-6-P glycans 8
ACCEPTED MANUSCRIPT (without derivatization) were not detected in our hands (data not shown) because the negative charge of M-6-P requires the use of a linear negative ion mode, which is insensitive compared with a positive ion mode. In contrast, all of 2-AA, 2-AB, and AA-Ac labeling
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increased the sensitivity and enabled the detection of M-6-P glycans (Fig. 2A). We found that the enhanced detection sensitivities of the bi-phosphorylated glycan differed depending on the fluorescent tag. The intensity of bi-phosphorylated glycan was normalized with the
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intensity of mono-phosphorylated glycan ([Intensity of bi-phsphorylated glycan]/[Intensity of mono-phosphorylated glycan]) and the highest value (1.6) was shown by 2-AA labeling,
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indicating the highest amount of bi-phosphorylated glycan (Fig. 2B). This is in good agreement with the HPLC result using 2-AA labeling as well as with the previous DNA sequencer-based analysis result [15]. In contrast, as a result of insensitive detection of biphosphorylated glycan, the normalized intensity values for the bi-phosphorylated glycan in
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the analyses with 2-AB and AA-Ac labeling were less than 1.0 at only 0.81 and 0.90, which would lead to an underestimation of bi-phosphoorylated glycan. Moreover, only 2-AA labeling allows simultaneous analysis of neutral and phosphorylated glycans because neutral
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glycans can be detected in a negative ion mode by the negative charge of 2-AA (Fig. 2A). These results clearly indicate the superior performance of 2-AA labeling for M-6-P glycan
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analysis using MALDI-TOF mass spectrometry. In this study, we showed that the optimization of elution solvent hydrophilicity is
necessary for HPLC analysis of M-6-P glycans with 2-AA labeling. After the optimization, 2AA labeling was superior for quantifying the content of bi-phosphorylated glycan compared with 2-AB and AA-AC labeling. Moreover, 2-AA labeling showed the highest sensitivity in detection of the bi-phosphorylated glycan in MALDI-TOF MS analysis.
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Acknowledgement This work was supported by the Next-Generation BioGreen 21 Program (PJ011078) of the
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Rural Development Administration in Korea, and the National Research Foundation of Korea
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[NRF-2013M3A9B6075888].
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ACCEPTED MANUSCRIPT Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis. Anal Chem 85 (2013) 7462-70. [19] J.Y. Kang, O. Kwon, J.Y. Gil, and D.B. Oh, Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags. Data in Brief (2016) submitted. [20] G.A. Baumbach, P.T. Saunders, F.W. Bazer, and R.M. Roberts, Uteroferrin has N-
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asparagine-linked high-mannose-type oligosaccharides that contain mannose 6-phosphate.
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Figure legends Fig. 1. HPLC analysis of the 2-AA-labeled M-6-P glycans. The 2-AA-labeled glycans obtained from Scmnn1∆och1∆/YlMPO1 were analyzed by amine column HPLC using either
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the generally used solvents (A) or the optimized solvents (B). After uncapping (lower panels), the glycan containing two M-6-Ps (indicated by the arrow) was only detected in the HPLC profile with the optimized solvent system (B). (C) 2-AA-, 2-AB-, and AA-Ac-labeled M-6-P
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glycans were analyzed using the same solvents optimized for 2-AA-labeling. (D) Relative contents (%) of neutral (Man7-9GlcNAc2, white bar), mono-phosphorylated (P-Man8GlcNAc2,
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gray bar), and bi-phosphorylated (P2-Man8GlcNAc2, black bar) glycans were calculated from the integrated peak areas. (E) For comparison of MAH conditions, the relative contents of biphosphorylated glycan (P2-Man8GlcNAc2) were obtained from MAH conditions using formic acid (gray bar) or hydrochloric acid (black bar). (F) The relative contents of bi-
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phosphorylated glycans were obtained from single (gray bar) or repeated MAHs (white bar). The bars represent the average of three replicated experiments. Asterisks indicate statistically significant differences (p < 0.05). Symbols used for glycans are those suggested
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by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). Green circle: mannose, blue square: GlcNAc, P: phosphate. Unidentified peaks are represented by *.
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(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. MALDI-TOF mass spectrometry analysis of M-6-P glycans. (A) Mass spectra of 2AA-, 2-AB-, and AA-Ac-labeled glycans were obtained. (B) Relative intensity ratios were calculated from [P2-Man8GlcNAc2 intensity]/ [P-Man8GlcNAc2 intensity]. Symbols are identical to those used in Fig. 1. 14
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