Sialic acid linkage-specific alkylamidation via ring-opening aminolysis (aminolysis-SALSA)

Hisatoshi Hanamatsu; Takashi Nishikaze; Jun-ichi Furukawa

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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

Sialic acid linkage-specific alkylamidation via ring-opening aminolysis (aminolysis-SALSA)

Hisatoshi Hanamatsu, Dr

Hokkaido Univ

Email: pj.ca.iadukoh.dem@ustamanah_h

Takashi Nishikaze, Dr

Shimadzu Corporation

Email: pj.oc.uzdamihs@zakihsin

Jun-ichi Furukawa, Dr

Hokkaido Univ

Email: pj.ca.iadukoh.dem@uruf_j

Corresponding author.

Created: September 29, 2021; Last Update: December 23, 2021.

Introduction

Sialic acids widely occur as glycoconjugates and are often present on the nonreducing ends of sugars via α2,3-, α2,6-, and α2,8-linkages as structural isomers. Thus, it is difficult to distinguish structural isomers with the same mass using mass spectrometry. Several unique derivatization methods for MS analysis have been developed to facilitate differentiation of sialyl-linkage isomers (1–3). Previously, we reported a one-pot glycan purification−derivatization method employing a newly developed sialic acid linkage-specific alkylamidation (SALSA) (4). We found that cleavage of intramolecular lactone derived from α2,3- and α2,8-linked sialic acid and its amidation proceed instantaneously via ring-opening aminolysis as shown in Figure 1 (5). Therefore, aminolysis-SALSA can provide a shorter reaction time and a simplified protocol.

Protocol

In this chapter, sialic acid linkage-specific derivatization via ring-opening aminolysis (Aminolysis-SALSA) will be described for analyzing glycosphingolipid (GSL)- and N-glycan (Figures 2 and 3). Furthermore, we will describe an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts in Figure 4 (6).

Materials

1.: MultiScreen Solvinert 0.45 μm of Low-Binding Hydrophilic Polytetrafluoroethylene (PTFE) filter plate (Millipore, Billerica, MA, Cat #MSRLN0410)
2.: Filter plate seal (Greiner Bio-One, Frickenhausen, Germany, Cat #J676060)
3.: BlotGlyco® bead suspension in water (10 mg/mL) (Sumitomo Bakelite Co., Ltd., Tokyo, Japan, Cat #BS-45408)
4.: 10 μM of A2GN1 (Neu5Ac2Gal2GlcNAc2 + Man3GlcNAc1) (Tokyo Chemical Industry, Tokyo, Japan, Cat #D4065)
5.: Acetonitrile (FUJIFILM Wako Pure Chemical, Osaka, Japan, Cat #018-19853)
6.: 2% Acetic acid (FUJIFILM Wako Pure Chemical, Cat #012-00245) in acetonitrile
7.: 2 M Guanidine hydrochloride (FUJIFILM Wako Pure Chemical, Cat #077-02435)
8.: 1% Triethylamine (FUJIFILM Wako Pure Chemical, Cat #202-02646) in methanol
9.: 10% Acetic anhydride (FUJIFILM Wako Pure Chemical, Cat #011-00271) in methanol
10.: 10 mM of Hydrochloric acid (FUJIFILM Wako Pure Chemical, Cat #192-02175)
11.: Methanol (FUJIFILM Wako Pure Chemical, Cat #134-14523)
12.: DMSO: Dimethyl sulfoxide (FUJIFILM Wako Pure Chemical, Cat #040-32815)
13.: SialoCapper™-ID Kit (Shimadzu Corporation, Kyoto, Japan, Cat# 225-42160-58 (JP/EN) 225-42160-46 (CN))
14.: Nα-((aminooxy)acetyl)tryptophanylarginine methyl ester (aoWR)
15.: Hydrophilic interaction liquid chromatography (HILIC) micro-elution plate (Waters, Milford, MA, Cat #186002780)
16.: HILIC pre-wash solution: 99% H₂O and 1% acetic acid
17.: HILIC wash solution: 95% acetonitrile, 4% H₂O, and 1% acetic acid
18.: HILIC loading solution: 99% acetonitrile and 1% acetic acid
19.: HILIC elution solution: 5% acetonitrile, 94% H₂O, and 1% acetic acid
20.: Matrix solution: 10 mg/mL of 2,5-dihydrobenzoic acid (Sigma-Aldrich, Cat #149357) in 30% acetonitrile

Instruments

1.: Micro Tube mixer (MT-400: TOMY, Tokyo, Japan)
2.: Vacuum manifold for 96-well plates (Waters)
3.: Ultraflex II TOF/TOF MS instrument (Bruker Daltonics, Bremen, Germany)
4.: Matrix-assisted laser desorption/ionization (MALDI) polished steel target plate (Bruker Daltonics, Cat #8280781)
5.: FlexAnalysis 3.0 software (Bruker Daltonics)

Methods

1.

Glycoblotting combined with Aminolysis-SALSA

a.: Dispense 5 mg of BlotGlyco® beads on a filter plate (Note 1).
b.: Set the filter plate into a vacuum manifold.
c.: Add up to 50 μL of prepared samples to each well of the filter plate.
d.: Add optimal volume of 10 μM of A2GN1 as an internal standard.
e.: Add 450 μL of 2% acetic acid in acetonitrile (Note 2).
f.: Incubate at 80°C for 60 min (Note 3).
g.: Set the filter plate into a vacuum manifold.
h.: Wash by adding 200 μL of 2 M guanidine hydrochloride and repeat twice (Note 4).
i.: Wash by adding 200 μL of H₂O and repeat twice.
j.: Wash by adding 200 μL of 1% triethylamine in methanol and repeat twice.
k.: Add 100 μL of 10% acetic anhydride in methanol and stand for 30 min (Note 5).
l.: Wash by adding 200 μL of 10 mM of hydrochloric acid and repeat twice.
m.: Wash by adding 200 μL of methanol and repeat twice.
n.: Wash by adding 200 μL of DMSO and repeat twice.
o.: Add 100 μL of iPA solution prepared from Reagents A and B in SialoCapper-ID Kit and gently shake by micro tube mixer for 1 h (Note 6).
p.: Wash by adding 200 μL of methanol and repeat twice.
q.: Add 100 μL Reagent C in SialoCapper-ID Kit and shake gently to ensure that the beads are immersed in the liquid (Notes 7 and 8).
r.: Wash by adding 200 μL of methanol and repeat twice.
s.: Wash by adding 200 μL of H₂O and repeat twice.
t.: Add 20 μL of 20 mM of aoWR (Note 9).
u.: Add 180 μL of 2% acetic acid in acetonitrile.
v.: Incubate at 80°C for 45 min.
w.: Add 100 μL of H₂O and elute aoWR-labeled glycans.

2.

aoWR-labeled Glycan Purification

a.: Wash the well of the HILIC micro-elution plate with 200 μL of HILIC prewash solution and repeat.
b.: Wash by adding 200 μL of HILIC wash solution and repeat.
c.: Add 450 μL of HILIC loading solution to 50 μL of the aoWR-labeled glycan sample (Step 1w) and transfer to the HILIC micro-elution plate.
d.: Wash by adding 200 μL of HILIC wash solution and repeat.
e.: Elute with 100 μL of HILIC elution solution.
f.: Analyze by MALDI-time of flight mass spectrometry (MALDI-TOF MS).

3.

MALDI-TOF MS Analysis

a.: Mix 0.5 μL of sample (Step 4e) and 0.5 μL of matrix solution on the target plate.
b.: Dry the spots on the target plate at room temperature.
c.: Load the target plate into the instrument and select the appropriate MS method.
d.: Run the instrument in reflector mode.
e.: Collect the MS data with 1,000–2,000 laser shots per sample and sum the collected data.
f.: Quantify the detected glycans using the area of the internal standard (Note 10).

Notes

1.: Cover the unused well with a plate seal and dispense 500 μL of BlotGlyco® bead suspension (10 mg/mL) into a well.
2.: Add nine times the amount of 2% acetic acid in acetonitrile to the filter plate well.
3.: Continue heating until the solvent is completely dry.
4.: After the last wash with various solvents, wipe the bottom of the filter plate with a paper towel.
5.: Freshly prepare 10% acetic anhydride in methanol.
6.: Add 970 μL of Reagent A to Reagent B (both in SialoCapper-ID Kit) and mix with vortex mixer (iPA solution). Reagent B is insoluble and should be mixed carefully (4,5).
7.: In this step, α2,3-linked sialic acid can be isotopically labeled with 2.9 M deuterated ethylamine hydrochloride dissolved in 10% tert-butylamine aqueous solution (6). Since tert-butylamine has a large steric hindrance, the aminolysis-SALSA reaction by this base will not proceed. O-acetylated sialic acids cannot be detected due to cleavage of O-acetyl group under the SALSA containing strong base condition.
8.: If using a 96-well filter plate for glycoblotting purification, refer to reference (5).
9.: aoWR was prepared as described previously (7). Labeling of the reducing end by deuterated aoWR is also possible. Moreover, liquid chromatography–mass spectrometry analysis is available by labeling the reducing end with 2-aminobenzamide (2-AB) (6).
10.: Although α2,8- and α2,3-linked sialic acid residues were derivatized with methylamine, these glycan isomers provided the different fragmentation patterns by MALDI-TOF/TOF analysis and could be distinguished.

References

1.: Reiding KR, Blank D, Kuijper DM, Deelder AM, Wuhrer M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification. Anal Chem. 2014 Jun 17;86(12):5784–93. [PubMed: 24831253] [CrossRef]
2.: Liu X, Qiu H, Lee RK, Chen W, Li J. Methylamidation for sialoglycomics by MALDI-MS: a facile derivatization strategy for both alpha2,3- and alpha2,6-linked sialic acids. Anal Chem. 2010 Oct 1;82(19):8300–6. [PubMed: 20831242] [CrossRef]
3.: Holst S, Heijs B, de Haan N, van Zeijl RJ, Briaire-de Bruijn IH, van Pelt GW, Mehta AS, Angel PM, Mesker WE, Tollenaar RA, Drake RR, Bovee JV, McDonnell LA, Wuhrer M. Linkage-specific in situ sialic acid derivatization for N-glycan mass spectrometry imaging of formalin-fixed paraffin-embedded tissues. Anal Chem. 2016 Jun 7;88(11):5904–13. [PubMed: 27145236] [CrossRef]
4.: Nishikaze T, Tsumoto H, Sekiya S, Iwamoto S, Miura Y, Tanaka K. Differentiation of sialyl linkage isomers by one-pot sialic acid derivatization for mass spectrometry-based glycan profiling. Anal Chem. 2017 Feb 21;89(4):2353–60. [PubMed: 28194959] [CrossRef]
5.: Hanamatsu H, Nishikaze T, Miura N, Piao J, Okada K, Sekiya S, Iwamoto S, Sakamoto N, Tanaka K, Furukawa JI. Sialic acid linkage specific derivatization of glycosphingolipid glycans by ring-opening aminolysis of lactones. Anal Chem. 2018 Nov 20;90(22):13193–9. [PubMed: 30335964] [CrossRef]
6.: Hanamatsu H, Nishikaze T, Tsumoto H, Ogawa K, Kobayashi T, Yokota I, Morikawa K, Suda G, Sho T, Nakai M, Miura N, Higashino K, Sekiya S, Iwamoto S, Miura Y, Furukawa JI, Tanaka K, Sakamoto N. Comparative glycomic analysis of sialyl linkage Isomers by sialic acid linkage-specific alkylamidation in combination with stable isotope labeling of alpha2,3-linked sialic acid residues. Anal Chem. 2019 Nov 5;91(21):13343–8. [PubMed: 31577134] [CrossRef]
7.: Uematsu R, Furukawa J, Nakagawa H, Shinohara Y, Deguchi K, Monde K, Nishimura SI. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis. Mol Cell Proteomics. 2005 Dec;4(12):1977–89. [PubMed: 16170054] [CrossRef]

Footnotes

: The authors declare no competing or financial interests.

Figures

Figure 1:

Analytical scheme of ring-opening-aminolysis-sialic acid linkage-specific alkylamidation (SALSA). The ring-opening-aminolysis reaction proceeds via cleavage of lactone derived from α2,3- and α2,8-linked sialic acids and their instantaneous and simultaneous amidation. Reprinted and modified with permission from (5).

Figure 2:

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) spectra showing GSL-glycans by various modifications of sialic acids. (A) GSL- glycans analyzed by conventional methods, including methyl esterification of sialic acid. (B) General sialic acid linkage-specific alkylamidation (SALSA) method for GSL-glycan analysis wherein α2,3- and α2,8-linked sialic acids are derivatized with methylamine. (C) GSL-glycans analyzed by the aminolysis-SALSA method. Reprinted and modified with permission from (5).

Figure 3:

Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) spectra of N-glycans of human serum by aminolysis- sialic acid linkage-specific alkylamidation (SALSA) method.

Figure 4:

Schematic illustration of comparative N-Glycomics using dual isotope labeling. Reprinted and modified with permission from (6).

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Bookshelf ID: NBK593836PMID: 37590587

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