User Benefits Application News Analysis of Serum N-Glycans of Gene-KO Mouse Using MALDI-7090 and SialoCapper-ID Kit  Sialic acid linkage isomer can be discriminated only by mass spectrometry without LC separation or sialidase enzyme treatment.  By combining with glycan purification beads "BlotGlyco®," glycan purification and sialic acid derivatization can be performed as a one-pot reaction.  By using the MS 2 analysis, the sialic acid linkage can be determined more reliably, omitting the verification by other biochemical experiments. M. Inuzuka, T. Nishikaze Sialic Acid Stabilizing Kit for Linkage Isomer Discrimination: SialoCapper™-ID Kit MALDI-TOF Mass Spectrometer: MALDI-7090  Introduction Protein glycosylation is involved in various biological events. In particular, sialic acid at the non-reducing end of the glycan is important because its presence and linkage type have been implicated in viral infections and cancer. Usually, liquid chromatography (LC) separation and sialidase treatment are used to discriminate sialic acid linkage isomers; however, there are technical problems such as the need for structurally unambiguous standard samples and the difficulty in discriminating complicated glycans. In recent years, sensitive and high-throughput mass spectrometry (MS) has been widely used for glycan analysis. However, there have been some intrinsic problems, such as the loss of sialic acid residues during analysis and the inability to distinguish linkage isomers with the same mass. In this article, we introduce an analysis example of N-glycans released from serum glycoproteins of sialyltransferase knock out (KO) mice. Samples were derivatized using the sialic acid stabilizing kit for linkage isomer discrimination “SialoCapper-ID Kit” followed by analysis using a MALDI-TOF-MS. Fig. 1 Derivatization scheme of SALSA method α2,6- and α2,3-linked sialic acids are amidated by isopropylamine (iPA) and methylamine (MA), respectively, by sequential two-step reactions.  Sialic Acid Linkage-Specific Derivatization Our patented sialic acid linkage-specific alkylamidation (SALSA method) prevents the loss of sialic acid residues during both glycan pretreatment and MS analysis by neutralizing the residues. In addition, it allows the MS-based discrimination of sialic acid linkage isomer by derivatizing the residues in a linkage-specific manner. 2,3) The SialoCapper-ID Kit is a novel reagent kit for glycan pretreatment that simplifies the SALSA procedure. O NH O H OH O H O OH O H H O O H O O H O N H O H OH OH C O C H3 O N H CH3 CH3 O NH O H OH O H O O O H O O H OH O OH N H O H OH O H C O C H3 O O NH O H OH O H O OH O H H O O H O O H O N H O H OH OH C O C H3 O OH O NH O H OH O H O OH O H O O H OH O OH C N H O H OH O H C O C H3 O OH α2,6-sialylation α2,3-sialylation -18 Da Lactone form O NH O H OH O H O OH O H O O H OH O OH C N H O H OH O H C O C H3 O NH CH3 O NH O H OH O H O OH O H H O O H O O H O N H O H OH OH C O C H3 O N H CH3 CH3 iPA derivative MA derivative （Δm = 0 Da） （Δm = 59 Da） （Δm = 28 Da） First reaction Second Reaction iPA derivative + 41 Da + 41 Da + 13 Da  Glycan Release from Glycoproteins First, serum glycoproteins derived from gene-KO mice were denatured and reduced with SDS and DTT. After adding NP-40, PNGaseF was added, and N-linked glycans (N-glycans) were released from the glycoprotein by reacting the solution at 37 o C for o/n (ca. 18 h). Fig. 2 Glycan purification and solid-phase sialic acid derivatization using BlotGlyco® and SialoCapper-ID Kit Fig. 3 MALDI-TOF mass spectrometer “MALDI-7090”  Sialyltransferase-KO Mouse Representative α2,6-sialyltransferases, ST6-Gal1 and ST6-Gal2 were knocked out. For comparison, following four types of mice were prepared; without KO (WT), ST6Gal1-KO, ST6Gal2-KO, and double KO (DKO).  N-Glycan Purification and Solid-phase Sialic Acid Linkage-Specific Derivatization Released N-glycans were purified using “BloGlyco®” (Sumitomo Bakelite Co., Ltd.). BlotGlyco® is a glycan purification bead that can specifically capture the reducing end of glycans. By using the beads in combination with the SialoCapper-ID Kit, glycan purification and sialic acid derivatization can be performed as a one-pot reaction (Fig. 2). BlotGlyco® BlotGlyco® Hz Hz Glycan capture BlotGlyco® BlotGlyco® BlotGlyco® BlotGlyco® Glycan release Sialic Acid Derivatization by SialoCapper-ID Kit  Glycan Labeling and Mass Spectrometry The reducing end of the N-glycan released from BlotGlyco® was labeled with 2-aminobenzoic acid (2AA), and excess reagents were removed using a commercially available HILIC-SPE chip. The sample solution and matrix solution were deposited on a MALDI plate and left to dry. Mass spectra were obtained using MALDI-7090 (Fig. 3) in the negative ion reflectron mode. Super- DHB was used for the matrix. The ASDF (Axial Spatial Distribution Focusing) function was used for MS 2 measurement. This allows high-resolution MS 2 spectra to be obtained even under high-energy CID conditions, enabling more accurate analysis.

Application News www.shimadzu.com/an/ Shimadzu Corporation Analytical & Measuring Instruments Division Global Application Development Center © Shimadzu Corporation, 2021 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.  Conclusion By using BlotGlyco® and SialoCapper-ID Kit, glycan purification and sialic acid linkage-specific derivatization can be easily performed as one-pot reaction. Although the sialic acid linkage type on the N-glycan treated with the kit can be discriminated by MS 1 analysis, MS 2 analysis is valuable for confirming unexpected results. In this study, we obtained the presence of α2,6-sialic acid, which is controlled by an enzyme other than ST6Gal1 and ST6Gal2. SialoCapper is a trademark of Shimadzu Corporation. BlotGlyco is a trademark of Sumitomo Bakelite Co., Ltd. First Edition: Apr. 2021 01-00110-EN  MS 1 Analysis of N-Glycan from Mouse Serum Various glycan peaks were detected with a difference of 28 Da corresponding to the difference of α2,3-/α2,6-linkages (Fig. 4). These m/z values were analyzed with the Software “Supporting Tool for SialoCapper-ID Kit” to calculate the monosaccharide composition (Table 1). Based on these results, we estimated the structure of N-glycans by considering past literatures and the known glycan biosynthetic pathways. By focusing on the di- antennary N-glycans, the disappearance of α2,6-sialylation in ST6Gal1-KO and DKO indicates that the enzyme responsible for the addition of α2,6-sialic acid is ST6Gal1. <References> 1) Ohmi Y. et al. (2020) Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6- sialyltransferase genes. Glycobiology in press 2) Nishikaze T, et al. (2017) Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling. Anal. Chem. 89: 2353–2360. 3) Hanamatsu H, et al. (2018) Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones. Anal. Chem. 90: 13193–13199.  Acknowledgments We would like to express our gratitude to Prof. Koichi Furukawa and Prof. Yuhsuke Ohmi of the Chubu University, for allowing us to analyze sera of sialyltranseferase-KO mice. (a) (b) (c) (d) % Intensity 0 20 40 60 80 100 m/z 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 2400.86 2546.96 2748.99 2080.85 2457.05 2429.01 2603.13 2108.88 2400.94 2080.83 2749.11 2547.04 2777.03 2603.00 3114.07 3114.19 3086.02 3086.17 2777.12 2456.94 2428.88 2108.78 ; Galactose (Gal) ; Mannose (Man) ; Fucose (Fuc) ; iPA-derivatized NeuGc (α2,6-linked) ; MA-derivatized NeuGc (α2,3-linked) ; GlcNAc 28Da 28Da 28Da To MS 2 28Da Fig. 4 MS 1 mass spectra of N-glycans derived from the glycoproteins of gene-KO mouse sera. (a) WT, (b) ST6Gal1-KO, (c) ST6Gal2-KO, (d) DKO m/z (calc.) Glycan Composition 2080.76 AA-Hex5HexNAc4NeuGc(a2,3-)1 2108.79 AA-Hex5HexNAc4NeuGc(a2,6-)1 2400.88 AA-Hex5HexNAc4NeuGc(a2,3-)2 2428.91 AA-Hex5HexNAc4NeuGc(a2,6-)1NeuGc(a2,3-)1 2456.94 AA-Hex5HexNAc4NeuGc(a2,6-)2 2546.94 AA-Hex5HexNAc4dHex1NeuGc(a2,3-)2 2603.00 AA-Hex5HexNAc4dHex1NeuGc(a2,6-)2 2749.03 AA-Hex5HexNAc4NeuGc(a2,6-)1NeuGc(a2,3-)2 2777.07 AA-Hex5HexNAc4NeuGc(a2,6-)2NeuGc(a2,3-)1 3086.14 AA-Hex6HexNAc5NeuGc(a2,3-)3 3114.17 AA-Hex6HexNAc5NeuGc(a2,6-)1NeuGc(a2,3-)2 Table 1 Monosaccharide Composition of N-glycans derived from mouse serum glycoproteins  MS 2 Analysis by High-Energy CID and ASDF The DKO sample lacking two different α2,6-sialyltransferases showed a peak at m/z 2749, suggesting the inclusion of α2,6- sialic acid residues. MS 2 analysis of this peak showed the neutral loss of 348 Da corresponding to isopropylamidated NeuGc (Fig. 5). This suggests the presence of α2,6-sialic acid, indicating that the α2,6-sialic acid is added by enzymes other than ST6Gal1 and ST6Gal2. m/z 2425 2426 2427 2428 2429 2430 2431 2432 2433 2434 2435 2436 2437 2438 2439 2440 2441 2429.25 m/z 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2748.99 2429.25 2401.20 2267.04 1395.56 2108.86 1760.74 1553.58 1233.44 1030.34 320 348 320 365 365 Fig. 5 MS 2 mass spectrum of m/z 2749 Supporting tool for SialoCapper-ID Kit is a software that can estimate glycan composition from given m/z values in the results of MS and deal with the mass change of sialic acid residues by sialic acid derivatization. Search condition: Mass Tolerance 0.2 Da Hex 3-10, HexNAc 2-10, dHex 0-1, Neu5Gc 0-4