2.1. Materials

The peptide:N-glycosidase F (PNGase F) was purchased from BioLabs (Ipswich, MA). The sialylglycopeptide (SGP, > 95 %) was purchased from FUSHIMI (Kagawa, Japan). The N-succinimidyl ferrocenecarboxylate was purchased from TCI (Japan). The cation exchange resin was purchased from The Nest Group (Southborough, MA, USA). All other reagents and solvents such as p-phenylenediamine, dimethylformamide (DMF), sodium cyanoborohydride (NaBH₃CN), hydrochloric acid (HCl), acetic acid, dimethyl sulfoxide (DMSO), anhydrous methanol, 50 % sodium hydroxide (NaOH), iodomethane (CH₃I), milli-Q-water, dichloromethane (DCM), formic acid, and acetonitrile (ACN) were purchased from SigmaAldrich (St. Louis, Missouri, US).

2.2. Synthesis of 4-aminophenyl ferrocenecarboxamide

The reaction of N-succinimidyl ferrocenecarboxylate with p-phenylenediamine is a conjugation reaction that forms a stable amide bond between carboxylate and amine. 100 mM N-succinimidyl ferrocenecarboxylate and 2 M p-phenylenediamine were dissolved in DMF to prepare stock solutions, respectively. Nsuccinimidyl ferrocenecarboxylate and p-phenylenediamine were transferred to a glass tube in an equivalent ratio of 1:20, mixed, and reacted. The reaction was performed at room temperature with vortexing every 20 min for 2 h. The purification of 4-aminophenyl ferrocenecarboxamide (APF) was performed by liquid-liquid extraction in which DCM was added to the reaction solution and then a 0.1 % formic acid aqueous solution was added and removed several times to remove the solvent and excess p-phenylenediamine.

2.3. Preparation of glycans

From the sialylglycopeptide (SGP) stock (2 mg/ mL) dissolved in water, 20 mg of SGP was taken into an Eppendorf tube and dried. After dissolving SGP in 1 × glycoprotein denaturing buffer, 1 munits of PNGase F was added and digested overnight at 37 °C to release biantennary sialic N-linked glycans. The solution was purified to the glycans through cation exchange and dried.

2.4. Permethylation of labeled glycans

The biantennary sialic N-linked glycans were conjugated with 4-aminophenyl ferrocenecarboxamide using a reductive amination. Permethylation of labeled glycans is as follows. The labeled glycans were resuspended in 200 µL dimethyl sulfoxide (DMSO) and 250 µL freshly dehydrated base (50 % NaOH in 2 mL of anhydrous DMSO). After sonication and vortexing under nitrogen, 100 µL iodomethane (CH₃I) was added and the mixture was vortexed vigorously for 10 min. 2 mL Milli-Q water was added to the labeled glycan sample and excess CH3I was removed by bubbling with a stream of nitrogen gas. After bubbling, the salt and remaining DMSO were removed by liquid-liquid extraction with water and DCM. The permethylated labeled glycans in DCM were dried under a nitrogen stream and stored at -20 °C for mass spectrometry analysis.

2.5. Analysis of glycans by LC-MS

The labeled glycans and synthesized labeling reagent were jointly analyzed using an LCQ Fleet mass spectrometer (Thermo Fisher Scientific, USA) at the Gyeongnam Bio and Anti-aging Core Facility Center for CWNU and the Chronic and Metabolic Diseases Research Center, Sookmyung Women’s University, equipped with an Accela 600 pump, Nano-flow Tee Splitter, and nano-electrospray ionization system. The glycan sample was resuspended in 50 µL of buffer B (0.1 % formic acid in 80 % acetonitrile) solution and 5 µL of the sample was injected for nanoLC-MS. Glycans were separated with a gradient of buffer A (0.1 % formic acid) and buffer B and ionization was performed by Nanospray Ion Source with a selfpacked C18 capillary column of a PicoTip^TM emitter (PicoFritTM SELF/P, 360 µm OD × 75 µm ID × 15 µm, New Objective) at 2.5 kV capillary voltage and 200 capillary temperature. Full MS spectra of glycans were collected in the mass range 150 ~ 2,000 m/z in positive ion and profile mode. MS/MS spectra of top 5 datadependent scans were obtained in centroid mode with settings of collision energy 38 %, isolation width 2 Da, and activation time 10 ms.

3.1. Preparation of ferrocene-based reagents for glycan labeling

Ferrocene-based derivatization is widely used in various fields of analytical chemistry, such as electrochemistry, atomic/molecular spectroscopy, and mass spectrometry combined with separation methods. The development of glycan derivatives with ferrocene is expected to provide various tools for the analysis of glycans. For this reason, 4-aminophenyl ferrocenecarboxamide (APF) was synthesized as a glycan labeling reagent for reductive amination by conjugation reaction of N-succinimidyl ferrocenecarboxylate and p-phenylenediamine as shown in Fig. 1.

Fig. 1(a) shows the synthesis of 4-aminophenyl ferrocenecarboxamide by nucleophilic attack of p-phenylenediamine on N-succinimidyl ferrocenecarboxylate, and its theoretical mass is shown to be 321.0690 Da (mono). After purification, the reagent was analyzed by LC-MS and was measured as [M+H]+ = 321.18 Da, indicating successful synthesis in Fig. 1(b). Fig. 1(c) shows the MS/MS spectrum of y ion fragments showing the cleavage of carbonyl-ferrocene bond of 186 Da and amide bond of 213 Da in the synthetic reagent.

3.2. Glycan labeling with 4-aminophenyl ferrocenecarboxamide

Various aromatic amines are commonly used as fluorescent derivatives of glycans for LC analysis and quantitative mass spectrometry by isotope-labeled derivatives of these. This approach is the derivatization of glycans by reductive amination using a newly synthesized ferrocene derivative, 4-aminophenyl ferrocenecarboxamide. Fig. 2 shows glycan labeling by reductive amination by conjugating the amine of 4-aminophenyl ferrocenecarboxamide to the aldehyde of the reducing end of free glycans to form a Schiff base and reducing it with a reducing agent (NaBH₃CN). The labeled glycans were purified by liquid-liquid extraction and prepared for mass spectrometry through permethylation for chemical stability and ionization efficiency.

Glycan labeling reagent, 4-aminophenyl ferrocenecarboxamide, is expected to be utilized as a versatile tool in glycan analysis. 4-Aminophenyl ferrocenecarboxamide contains both ferrocene and aromatic groups, which may contribute to its potential as a fluorophore or chromophore. Due to these structural features, it is being investigated for possible applications in areas such as electrochemistry, atomic analysis, and mass spectrometry. Additionally, this reagent is expected to be useful for purifying and separating glycans of various hydrophilic structures due to increased hydrophobicity.

3.3. Analysis of APF-labeled glycans by LCMS

Free biantennary sialic N-linked glycan released from SGP was labeled with 4-aminophenyl ferrocenecarboxamide. Unlabeled or labeled biantennary sialic N-linked glycans were separated by liquid chromatography and analyzed by mass spectrometry. Glycans were eluted from high buffer B by the gradient, and unlabeled glycans were eluted at 58 min and labeled glycans at 63 min, respectively, showing separation with a retention time of 5 min due to hydrophobicity. Fig. 3 shows the full mass spectra of unlabeled and labeled glycans. Glycans were analyzed as charge states of both divalent and trivalent cations by protonation. As shown in Fig. 3(a) and (b), the mass spectrum of the unlabeled glycan and the labeled glycan was obtained with a mass of [M +2H]²⁺ = 1385.50 Da and [M +2H]²⁺ = 1551.58 Da, respectively. The mass spectrum of APF-labeled glycans shows that APF is stably labeled and detected on glycans during purification, permethylation, and ionization for mass spectrometry.

Fig. 4 shows the MS/MS spectrum of 1551.58 Da (2+) for structural interpretation of glycans by fragmentation of APF-labeled glycans. In the fragmentation pattern, APF is structurally rigid, while the fragmentation of glycans is rich to facilitate structural interpretation of glycans. In general, dissociation of glycans appears as doubly charged ions, and dissociation of APF and glycans shows rich fragmentation from singly charged ions.