A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome

Analytica Chimica Acta xxx (xxxx) xxx

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A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome Yan Cai a, Ying Zhang a, b, Wenjuan Yuan c, Jun Yao a, Guoquan Yan a, Haojie Lu a, b, * a

Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, PR China Department of Chemistry and NHC Key Laboratory of Glycoconjugates Research, Fudan University, Shanghai, 200032, PR China c Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, PR China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Thiazolidine chemistry was for the first time extended to N-glycoproteome enrichment.
Covalent coupling time was shortened within 30 min without addition of toxic catalyst or sample-destroying reducing agent.
The new method showed great selectivity (1:100), high sensitivity (low fmol) and good reproducibility (CV<26%).
Cys-terminated magnetic nanoparticles was facile prepared by onestep modification of commercial amine magnetic beads.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 September 2019 Received in revised form 15 November 2019 Accepted 1 December 2019 Available online xxx

For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome. With the use of facile synthesis of Cys-terminated magnetic nanoparticles, the oxidized glycan moieties on glycopeptides could be selectively captured by the b-amino thiols groups on the surface of magnetic nanoparticles through thiazolidine formation. The coupling could be achieved within 30 min under mild condition, eliminating the addition of toxic catalyst or sample-destroying reducing agent. Also, the great enrichment performance for N-glycopeptides were obtained in terms of sensitivity (low fmol levels), selectivity (extracting Nglycopeptides from the mixture of glycopeptides and non-glycopeptides at a 1:100 molar ratio) and reproducibility (CVs<26%). Finally, this proposed method was successfully demonstrated by analyzing the N-glycoproteome from 2 mL human serum, which offers an alternative purification method for analysis of N-glycoproteome from complex biological samples. © 2019 Elsevier B.V. All rights reserved.

Keywords: Thiazolidine chemistry N-Glycoproteome Mass spectrometry Enrichment Ultrafast

* Corresponding author. Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, PR China. E-mail address: [email protected] (H. Lu). https://doi.org/10.1016/j.aca.2019.12.001 0003-2670/© 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Y. Cai et al., A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of NGlycoproteome, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.001

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1. Introduction N- glycosylation is one of the most frequent and structurally diverse post-translational modifications of eukaryotic proteins. Protein N-glycosylation plays critical roles in a wealth of biological process including protein folding, cell signaling, and the immune response [1,2]. Aberrant N-glycosylation is strongly associated with a variety of diseases such as tumor growth and metastasis, inflammation and rheumatoid arthritis [3]. Also, the majority of FDA-approved therapeutic antibodies are recombinant N-glycoproteins [4]. Therefore, the rapid and large-scale analysis of Nglycoproteins is a requirement for the discovery of novel clinical biomarkers and drug development. Currently, mass spectrometry (MS) has become a powerful tool for comprehensive N-glycoproteomic analyses [5,6]. However, Nglycopeptides analysis by MS is commonly hampered by their low abundance, inherent heterogeneity and suppression by numerous non-glycopeptides. Therefore, for in-depth N-glycoproteomic studies, selective enrichment of N-glycopeptides/N-glycoproteins prior to MS analysis is a critical step to reduce the complexity of biological samples [7]. To date, three main strategies are reported for N-glycopeptides enrichment: (1) enrichment based on lectin affinity/immunoaffinity (2) enrichment based on hydrophilic interaction (HILIC) (3) enrichment based on covalent interactions [8e10]. Among them, covalent capture is thought to be universal, unbiased and highly specific. Typically, boronic acid chemistry is widely used for the global analysis of glycopeptides. Recently, a new method using dendrimer-conjugated benzoboroxole was developed to enhance the glycopeptide enrichment by both synergistic and reversible covalent interactions between benzoboroxole and glycan [11]. Hydrazide chemistry is another prevalent covalent coupling for glycopeptides enrichment, primarily due to its high selectivity and commercial availability. However, this method commonly requires long coupling time (over 12 h) and an additional desalting step [12e14]. For this reason, reductive amination-based covalent coupling was introduced to N-glycopeptide enrichment, which significantly shortened coupling time to 4 h and eliminated the desalting step [15]. To further address the problem of side reaction between amino groups on peptides and oxidized aldehydes from N-glycopeptide, reductive amination combined with dimethlyation labeling was developed. The N-termius and side chain amino groups of peptides were blocked via dimethlyation labeling, thereby improving identification of Nglycosylation sites [16]. For reductive amination reaction, a reducing agent NaBH3CN was required to form stable CeN bond, which inevitably caused sample loss or damage. Therefore, anlinefunctionalized magnetic nanoparticles were synthesized and applied to enrich N-glycopeptides by nonreductive amination coupling, which eliminated the reducing agent and improved the enrichment sensitivity [17]. To further shorten coupling time and search for mild coupling reaction, oxime click chemistry was described for N-glycopeptide enrichment. For simple model glycoproteins, excellent enrichment performance was achieved within 1 h, together with improved sensitivity (fmol), specificity (capture glycopeptides from the mixture of glycopeptide and nonglycopeptides at a 1:100 M ratio) and reproducibility (CV<20%). For complex human serum sample, the coupling time of 4 h was still needed. Additionally, oxime click reaction was carried out under pH 4.5 and catalyzed by 100 mM anline [18]. Overall, enrichment methods based on covalent coupling for MS-based Nglycoproteomics still suffer from several problems such as long coupling time and harsh coupling conditions. Therefore, it is imperative to search for alternative reactions that would be rapid and mild for high-throughput N-glycoproteome profiling. Thiazolidine chemistry has been of particular interest for

decades in different areas, for instance, large scale cysteinecontaining peptides analysis, synthesis of peptide-oligonucleotide conjugates and construction of homogeneous biopharmaceuticals antibody-drug conjugates [19e22]. Essentially, thiazolidine chemistry is a nucleophilic addition-elimination reaction between aldehyde group and b-amino thiols to form thiazolidine ring (Fig. 1a). The main advantages of thiazolidine formation reaction are present as follows: (1) the reaction is very rapid due to the presence of sulfhydryl group with high nucleophilic reactivity. (2) The reaction condition is quite mild without addition of any toxic catalyst or sample-destroying reducing agent. (3) Compared to hydrazones and oximes, thiazolidines are more stable aldehyde derivatives [23e25]. These unique advantages inspire us to design and prepare a solid support bearing b-amino thiol, and then explore a solid-phase extraction method based on thiazolidine chemistry for rapid and highly efficient enrichment of N-glycoproteome. In this work, a thiazolidine formation-based method was developed for ultrafast and highly efficient solid-phase extraction of N-glycoproteome. As illustrated in Fig. 1b, after the oxidation of glycan moiety, both N-glycopeptides and O-glycopeptides could be selectively captured on the Cys-terminated magnetic nanoparticles through thiazolidine linkage between aldehyde groups from oxidized glycopeptides and b-amino thiols from solid support, but only N-glycopeptides could be specifically released via the use of PNGase F. Firstly, thiazolidine formation reaction was further optimized. Next, we prepared Cys-terminated magnetic nanoparticles as solid support by one-step modification. Finally, a thiazolidine formation-based method was developed for ultrafast and highly efficient solid-phase extraction N-glycoproteome. To our knowledge, this is the first proof of concept study where thiazolidine chemistry was demonstrated as a covalent coupling reaction in the field of N-glycoproteome enrichment. 2. Experimental section 2.1. Materials and chemicals Asialofetuin from fetal calf serum (ASF), myoglobin from horse heart (MYO), trypsin from bovine pancreas (TPCK treated), sodium acetate (NaAc), sodium periodate (NaIO4), sodium sulfite (Na2SO3), dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), N,N-diisopropylethylamine (DIEA), Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a-cyano-4-hydroxycinnamic acid (CHCA), formic acid (FA) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. N-(tert-Butoxycarbonyl)-S-trityl-Lcysteine (Boc-Cys(Trt)-OH), triethylsilane (Et3SiH), 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole monohydrate (HoBt) and LCysteine Methyl Ester Hydrochloride (CysOMe, 99%) were purchased from Tokyo Chemical Industry Co., Ltd. Ammonium acetate (NH4Ac, HPLC grade) was purchased from Roe Scientific Inc. Acetonitrile (ACN, LC/MS grade) was purchased from Fisher Chemical. Dichloromethane (DCM), dimethylformamide (DMF) and ethanol were obtained from Sinoreagent Chemical Reagent Co. Ltd. Amino-functionalized Fe3O4 nanoparticles (5 mg/mL) were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. The glycerol free peptide-N-glycosidase (PNGase F, 500 units/mL) was purchased from New England Biolabs. Sep-Pak C18 columns were obtained from Waters Corporation. Ultra-pure water was obtained from a Milli-Q Water System (Millipore). 2.2. Preparation and characterization of Cys-terminated magnetic nanoparticles According to the previous work with some modification, Cys-

Please cite this article as: Y. Cai et al., A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of NGlycoproteome, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.001

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Fig. 1. Illustration of solid-phase extraction of N-glycopeptides based on thiazolidine chemistry. (a) Thiazolidine formation between aldehyde group and b-amino thiols. (b) Selective solid-phase extraction of N-glycopeptides through thiazolidine formation. (c) The workflow of N-glycopeptides enrichment followed by MS analysis using Cys-terminated magnetic nanoparticles.

terminated magnetic nanoparticles were synthesized (Fig. 3a) [24]. Briefly, amino-functionalized Fe3O4 nanoparticles (32 mg, 31 mmol/ g) were washed 3 times with ethanol and 3 times with DMF. BocCys(Trt)-OH (148 mg, 0.32 mmol), PyBOP (125 mg, 0.24 mmol) and HOBt (32 mg, 0.24 mmol) were dissolved in DMF (4 mL) containing DIEA (62 mL, 0.36 mmol). The solution was mixed with amino-modified Fe3O4 nanoparticles and the conjugation was conducted at room temperature for 2 h. Afterwards, the magnetic nanoparticles were washed 3 times with DMF, 3 times with DCM and stored wet at 4  C. Prior to N-glycopeptides enrichment, deprotection of Boc and Trt groups was performed with TFA-DCM (1:1, v/v) in the presence of excessive Et3SiH at room temperature for 1 h. The nanoparticles were sequentially washed 2 times with DCM, 2 times with ethanol and 2 times with 50%ACN. Scanning electron microscope (SEM) images were obtained by a

scanning electron microscope (FEI) at an accelerating voltage of 10 kV. Samples dispersed at an appropriate concentration were cast onto a glass sheet at room temperature and sputter coated with gold. Transmission electron microscope (TEM) images were obtained by a JEM 2011 transmission electron microscope (JEOL) at an accelerating voltage of 200 kV. Samples dispersed at an appropriate concentration were cast onto a carbon-coated copper grid. Fouriertransformed infrared spectroscopy (FTIR) was performed on a Nicolet iS10 spectrometer (Thermo Fisher Scientific). The samples were dried and then mixed with KBr, followed by compressed to form pellets. The saturation magnetization curves were obtained by a MPMS3 magnetometer (Quantum Design) at 300 K. Zetasizer Nano-ZS (Malvern Panalytical) was used for the measurements of the zeta potential of the magnetic nanoparticles. The amount of sulfhydryl groups on Cys-terminated magnetic nanoparticles was

Please cite this article as: Y. Cai et al., A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of NGlycoproteome, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.001

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Fig. 2. Optimization of thiazolidine formation between aldehyde groups and b-amino thiols. MALDI-TOF mass spectra of standard peptide TEALEQGGLPK (a, c and e) and SIPPGLVNGLALQLR (b, d and f). (a) (b) native, (c) (d) aldehyde groups generation by 2 mM NaIO4 oxidation, (e) (f) addition of CysOMe through thiazolidine formation. (g) Generation of aldehyde groups at Ser and Thr ending peptides by oxidation.

Fig. 3. (a) Synthesis routine of Cys-terminated magnetic nanoparticles. (b) TEM image of Cys-terminated magnetic nanoparticles, the scale bar is 50 nm. (c) FTIR spectra and (d) magnetic hysteresis curves of (i) amino-functionalized Fe3O4 nanoparticles (ii) modification with Boc-Cys(Trt)-OH (iii) Cys-terminated magnetic nanoparticles.

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quantitated by Ellman Test. 2.3. Preparation of tryptic digests For standard proteins, ASF and MYO were dissolved in 20 mM ABC (pH ¼ 8.0) at a concentration of 1 mg/mL and denatured at 100  C for 5 min, respectively. After cooling to room temperature, trypsin was added to each solution at an enzyme-to-protein ratio of 1:40 (w/w). Then digestion of proteins were performed at 37  C overnight, followed by boiling to terminate the reaction. The proteins digest were lyophilized and stored at 20  C for further use. For human serum sample, it was kindly provided by Fudan University Shanghai Cancer Center. The research complied with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Shanghai Cancer Center. 2 mL of human serum was diluted with 198 mL of 20 mM ABC (pH ¼ 8.0) and denatured at 100  C for 5 min. After cooling to room temperature, the solution was treated with 10 mM DTT at 56  C for 30 min and alkylated with 20 mM IAA at room temperature for 1 h in the dark. Next, the solution was diluted with 200 mL of 20 mM ABC (pH ¼ 8.0). Trypsin was added at an enzyme-to-protein ratio of 1:30 (w/w), followed by proteins digestion at 37  C for 16 h. The reaction was quenched by the addition of TFA (final concentration, 0.1%), and then the tryptic digest were desalted using the Sep-Pak C18 columns. The eluted peptides were lyophilized and stored at 20  C for further use. 2.4. Enrichment of N-glycopeptides with Cys-terminated magnetic nanoparticles The lyophilized peptides were redissolved in 100 mM NaAc buffer (pH 5.5). Then, the cis-diols of the glycan moiety of the glycopeptides were oxidized by 10 mM NaIO4 at room temperature for 1 h in the dark. To quench the oxidation, Na2SO3 was added (final concentration, 20 mM) and the solution was incubated at room temperature for another 10 min. For buffer exchange, the oxidized peptides were lyophilized, redissolved with coupling solution and then incubated with Cys-terminated magnetic nanoparticles. To remove non-glycopeptides and other impurities, magnetic nanoparticles were sequentially washed with coupling solution, 50%ACN/0.1%TFA and 80%ACN/0.1%TFA with the help of a magnet. Afterwards, the magnetic nanoparticles were dispersed in 10 mM ABC (pH ¼ 8.0), followed by addition of 1 mL of PNGase F. The solution was incubated at 37  C overnight to release the glycopeptides from the magnetic nanoparticles. The supernatant containing deglycosylated glycopeptides was collected by magnetic separation and lyophilized for further MS analysis. 2.5. MS analysis and data processing All MALDI MS analysis were performed in positive ion reflection mode on a 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) equipped with a Nd:YAG laser (355 nm), a repetition rate of 400 Hz, and an acceleration voltage of 20 kV. Laser intensity was set to around 6000 to ensure good signal intensity without generation of extensive noise. 1000 laser shots were accumulated for each spectrum. CHCA matrix was prepared at a concentration of 5 mg/mL in 50%ACN (v/v) containing 0.1%TFA. MYO tryptic digests was used as internal standard to calibrate the MALDI mass spectrometer. The nano LC-MS/MS analysis was performed on a UltiMate 3000 UPLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with an online nanoelectrospray ion source (Thermo Fisher Scientific). The lyophilized deglycosylated glycopeptides were resuspended with buffer A

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(water with 0.1%FA). The sample solution was loaded onto the trap column (Thermo Fisher Scientific, Acclaim PepMap C18, 100 mm  2 cm) at a flow rate of 10 mL/min for 3 min. Subsequently, peptides were separated on the analytical column (Thermo Fisher Scientific, Acclaim PepMap C18, 75 mm  15 cm) at a flow rate of 300 nl/min with a linear gradient of buffer B (ACN with 0.1%FA) from 2 to 40% over 70 min. The electrospray voltage was set at 2.1 kV and the temperature of ion transfer tube was set at 275  C. A data-dependent acquisition (TopN) MS mode was used. For each cycle of duty, it consisted of one full-MS survey scan (350e1800 m/ z) with a resolution of 60 K. Then MS/MS scans with a resolution of 15 K were conducted for ten of the most abundant precursor ions. For MS, the automatic gain control (AGC) was set to 400000 ions, with maximum accumulation times of 50 m s. For MS/MS, precursor ions were activated using 30% normalized collision energy, an isolation window of 1.6 m/z and the AGC was set to 50000 ions, with maximum accumulation time of 50 m s. Single charge state was rejected and dynamic exclusion was used with one microscan and 60 s exclusion duration. Thermo Proteome Discoverer software (Version 2.1) with the SEQUEST search engine and pFind software (Version 3.0) were used for database searching. All acquired MS/MS spectra were searched against human database (Release 2018-06-12, 20293 sequences) downloaded from UniProtKB. The search parameters were set as follows: fully tryptic digestion, up to two missed cleavages, fixed modifications: carbamidomethylation of cysteine (þ57.02 Da), variable modifications: oxidation of methionine (þ15.99 Da), acetylation of protein N-terminal (þ42.01 Da) and deamidation of asparagine (þ0.98 Da), 10 ppm mass tolerance for precursor ions, 0.05 Da mass tolerance for product ions, peptide charge: þ2, þ3, þ4. The target-decoy method was employed. The percolator algorithm was used to control peptide level false discovery rates (FDR) lower than 1%. Only peptides sequence containing N-X-S/T motif (XsP) were considered as N-glycopeptides. 3. Results and discussion 3.1. Optimization of thiazolidine chemistry A thiazolidine formation-based approach for N-glycopeptides enrichment involves in the coupling of aldehyde groups from oxidized N-glycopeptides to b-amino thiols on Cys-terminated magnetic nanoparticles (Fig. 1b). Thus, to obtain the highest coupling yield within shortest possible time, we further investigated and optimized thiazolidine formation reaction. Two standard peptides TEALEQGGLPK (monoisotopic mass: 1141.60, Fig. 2a) and SIPPGLVNGLALQLR (monoisotopic mass: 1546.92, Fig. 2b), as well as CysOMe (monoisotopic mass: 135.04) bearing b-amino thiol structure were chosen. At first, 2 mM NaIO4 treatment was performed to generate aldehyde groups at the N-termius of both peptides (Fig. 2g), which caused desired 45 Da (Fig. 2c) and 31 Da (Fig. 2d) mass decrease for TEALEQGGLPK and SIPPGLVNGLALQLR, respectively [26,27]. Subsequently, influential parameters of thiazolidine chemistry including reaction temperature, pH, organic solvent content and reaction time were systematically optimized according to the previous reports [23,28]. Coupling yield is calculated by dividing peak intensity of total peak intensities of thiazolidine product by the total peak intensities of residual reactant and thiazolidine product. As shown in Fig. S1a, reaction temperature was critical to the yield. As the reaction temperature reached 50  C or above, over 95% yield could be obtained for both peptides. Thiazolidine formation was not very sensitive to pH across the range 4.5e6.8, thereby allowing the coupling reaction with high yield using NH4Ac buffer at close to neutral pH (Fig. S1b). Addition of different ACN content had no significant effect on the yield for

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TEALEQGGLPK peptide and slightly improved effect on the yield for SIPPGLVNGLALQLR peptide. Considering subsequent sample solubility and minimized nonspecific absorption, 50%ACN was selected (Fig. S1c). Remarkably, almost complete conversion could be achieved within only 30 min coupling time (Fig. S1d). This unique feature was attributed to the sulfhydryl group with high nucleophilic reactivity as mentioned above. In addition, a large excess of CysOMe was used (data not shown) and 5 mM TCEP was applied to avoid disulfide bonds formation. In conclusion, the coupling reaction could be carried out under mild condition: 10 mM NH4Ac buffer (pH 6.8) containing 50%ACN and 5 mM TCEP at 50  C for 30 min. Under optimal condition, as shown in Fig. 2e and f, the expected mass increase of 117 Da for oxidized TEALEQGGLPK (m/z 1214.49) and oxidized SIPPGLVNGLALQLR (m/z 1633.65) were observed, respectively. The original signals of oxidized peptides were hardly seen, indicating almost complete (>96%) addition of CysOMe at oxidized N-termius of both peptides through thiazolidine formation. The high coupling yield within such a short time would make thiazolidine chemistry potentially useful for ultrafast and highly efficient N-glycopeptides enrichment. 3.2. Preparation and characterization of Cys-terminated magnetic nanoparticles As shown in Fig. 3a, the design and synthesis of Cys-terminated magnetic nanoparticles is quite simple and straightforward, which involves one-steps modification of the readily accessible aminofunctionalized Fe3O4 nanoparticles with Boc-Cys(Trt)-OH by classic condensation reaction between amines and carboxylic acids. Prior to glycopeptides enrichment, the protective groups of Trt and Boc would be removed by 50%TFA, generating the active b-amino thiols. The as-prepared Cys-terminated magnetic nanoparticles were characterized by different techniques. The shape, size, and morphology were observed by SEM and TEM. As shown in SEM images (Fig. S2), the Cys-terminated magnetic nanoparticles maintained uniform morphology with little difference in size and shape from amino-functionalized Fe3O4 nanoparticles. The TEM image indicated Cys-terminated magnetic nanoparticles possessed spherical shape with a diameter of approximately 110 nm (Fig. 3b). Moreover, the successful modification on the nanoparticles was validated by FTIR spectra (Fig. 3c). As expected, the adsorption peaks at 586 cm1 and at 1087 cm1 indicated the stretching vibration of the FeeO and the SieOeSi. The broad peak at 3490 cm1 was assigned to NeH stretching vibration. After modification with Boc-Cys(Trt)-OH, the typical vibrations of Boc group were observed at 1154 cm1 (C(CH3)3 deformation), 1730 cm1 (C]O stretching vibration) and 2960 cm1 (CeH stretching vibration). After deprotection with TFA, the peaks at 1154 cm1 and 1730 cm1 disappeared while the peak at 1645 cm1 (amide C]O stretching vibration) was still observed. The above results indicated the successful grafting of Boc-Cys(Trt)-OH onto the amino-functionalized Fe3O4 nanoparticles and the successful removal of both Trt and Boc protective group. Considering the band at 2550 cm1 attributed to SeH was weak here, the zeta potential measurements were performed to further support the results of FTIR spectra. Due to the reduced surface charge by the success introduction of Boc-Cys(Trt)OH, the zeta potential of magnetic nanoparticles initially decreased from 17.4 mV to 6.1 mV. Subsequently, the zeta potential increased up to 19.7 mV because TFA deprotection regenerated amino and sulfhydryl groups (Fig. S3). Furthermore, the amount of sulfhydryl groups immobilized on magnetic nanoparticles was estimated to be 26 mmol/g by Ellman Test, indicating a high overall grafting efficiency (~84%). In addition, the VSM results showed that the Cysterminated magnetic nanoparticles had superparamagnetic

behavior with a VSM of 70 emu/g (Fig. 3d), making it easily separated from and re-dispersed into the liquid phase within 20 s with the aid of a magnet (Fig. S4). This great magnetic response and redispersibility would facilitate a rapid and efficient solid-phase extraction of N-glycopeptides.

3.3. Ultrafast and highly efficient enrichment of N-glycopeptides from standard glycoproteins To demonstrate the feasibility of thiazolidine chemistry into Nglycopeptides enrichment, a model N-glycoprotein ASF with three known N-glycosylation sites were tested using Cys-terminated magnetic nanoparticles. The typical enrichment workflow was shown in Fig. 1c. ASF digests were firstly oxidized to generate aldehydes, and then incubated with the Cys-terminated magnetic nanoparticles. The b-amino thiols on the nanoparticles exclusively reacted with aldehydes from the oxidized glycopeptides, resulting in covalent coupling of glycopeptides to the nanoparticles. After washing of other unbound peptides, the N-glycopeptides were subsequently released by PNGase F for MS analysis. As shown in Fig. 4a, when ASF tryptic digests were directly analyzed without enrichment, the spectrum was dominated by non-glycopeptides, and glycopeptides were severely suppressed with only one Nglycopeptide detectable at m/z 5004.2 with very low signal-tonoise (S/N) ratio. In contrast, after thiazolidine-mediated enrichment followed by PNGase F release, seven dominate peaks of ASF deglycosylated glycopeptides were detected with a clean background in the spectrum (Fig. 4b). The peaks observed at m/z 1627.7, 1754.8, 1780.8, 1933.8, 1951.8, 3017.5, and 3558.8 represented ASF deglycosylated glycopeptides and corresponding fragments, which covered all of the three known N-glycosylated sites of ASF at N99CS, N176GS and N156DS. The detailed sequence information of each deglycosylated glycopeptide were listed in Table S1. The results demonstrated that thiazolidine chemistry could be successfully applied to N-glycopeptides ultrafast enrichment (within 30 min) without addition of any toxic catalyst or sample-destroying reducing agent. To further investigate the selectivity of this method in relatively complex samples, the mixture of ASF tryptic digests and MYO (a

Fig. 4. MALDI-TOF mass spectra of ASF tryptic digests. (a) Direct analysis without enrichment (b) after enrichment with Cys-terminated magnetic nanoparticles followed by PNGase F release. The symbol “^” indicates glycopeptides. The symbol “C” indicates non-glycopeptide. The symbols “@”, “*” and “#” indicate deglycosylated glycopeptides (three glycosylation sites) and their corresponding fragments.

Please cite this article as: Y. Cai et al., A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of NGlycoproteome, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.001

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standard non-glycoprotein) tryptic digests at a series of molar ratios 1:1, 1:10 and 1:100 were selectively enriched with Cysterminated magnetic nanoparticles. As seen in Fig. S5a, before enrichment, none of ASF glycopeptides could be detected due to the severe interference of numerous non-glycopeptides from MYO (for instance, at a molar ratio 1:100). On the contrary, after enrichment, all peaks of deglycosylated glycopeptides could be easily observed with a clean background in the mass spectra (Figs. S5be5d). The results demonstrated the glycopeptides could be fished out from the relatively complex mixture by Cys-terminated magnetic nanoparticles and thiazolidine chemistry-based solid-phase extraction method showed great enrichment selectivity for Nglycopeptides. Also, the proposed method showed other good enrichment performance in terms of sensitivity, reproducibility and recovery. First, a series of diluted ASF tryptic digests were oxidized and captured by Cys-terminated magnetic nanoparticles. The lowest detectable concentration was found to be at 20 ng/mL (Fig. S6), indicating the detection limit was estimated to be approximate 0.4 ng/mL (low fmol levels) in terms of representative deglycosylated glycopeptide LCPDCPLLAPLNDSR. Second, this method had good enrichment reproducibility. Selective enrichment of N-glycopeptides from ASF tryptic digests were performed in sixplicates. As shown in Table S2, the CVs of S/N ratios of deglycosylated glycopeptides and corresponding fragments were calculated in the range from 4.1% to 25.7%. Third, the recovery of this method was evaluated by stable isotope dimethyl labeling according to the previous work [31]. It was found that the recovery was around 71.4%, which was comparable to our previously developed oxime click chemistry method (Fig. S7) [18]. The good enrichment performance of this method was probably due to (1) the relatively mild coupling condition without addition of toxic catalyst or sampledestroying reducing agent, eliminating the sample destroy or loss; (2) the great magnetic properties, allowing ease of use and rapid solid-liquid separation. Collectively, these results make us believe that thiazolidine formation-based method is a powerful tool for ultrafast and highly efficient enrichment of Nglycopeptides.

glycoproteins and glycopeptides was listed in Table S3. The result showed comparable performance to several recent HILIC-based reports [30e33] (Table S4) and better performance than the previous covalent interactions-based reports [15,17,18] (Table S5), indicating our approach a competitive method for analysis of Nglycoproteome from complex biological samples.

3.4. Ultrafast and highly efficient enrichment of N-glycoproteome from human serum

Acknowledgement

Serum glycoproteome profiles are of great significance for the discovery of clinical biomarker candidates and novel drug development. However, the 10 most abundant proteins represent approximately 90% of the total protein mass in human serum [29]. Therefore, the comprehensive analysis of serum glycoproteome without high-abundance proteins depletion is particularly challenging. To evaluate the feasibility of the proposed method in analysis of this complex biological sample, 2 mL of normal human serum provided by Fudan University Shanghai Cancer Center was tested. After reduction and alkylation, the serum sample was digested into peptides by trypsin, followed by oxidation, selectively enriched with Cys-terminated magnetic nanoparticles. The deglycosylated glycopeptides were released by PNGase F and subjected to nano LC-MS/MS analysis. The identification of N-glycopeptides and N-glycosylation sites could be unambiguously assigned by considering both the consensus motif of N-X-S/T (XsP) and the mass shift of 0.98 Da of asparagine (N) converted into aspartic acid (D). A total of 240 N-glycosylation sites were identified in 223 unique glycopeptides corresponding to 115 glycoproteins in three biological replicates (Fig. S8). The high overlap (79%) of identified glycoproteins indicated the good reproducibility of our method. The specificity of the identified glycoproteins could be approximately 70%. The detailed information of the identified

4. Conclusion In summary, we developed a novel N-glycopeptides enrichment method using a Cys-terminated magnetic nanoparticles. To our knowledge, it is the first time that thiazolidine chemistry is extended to large-scale MS profiling of N-glycoproteins, and definitely provides an ultrafast, highly efficient and easily accessible protocol. It should be noted that besides glycopeptides, Ser or Thr ending peptides would also undergo thiazolidine chemistry. But only deglycosylated glycopeptides could be released from the beads by PNGase F and further identified by MS, whereas Ser or Thr ending peptides captured could not be “eluted” and interfere with the subsequent MS analysis. The limitation of the approach is, like covalent hydrazide chemistry enrichment, only peptide backbone of glycopeptides are released by PNGase F for MS analysis while glycans are oxidized and then coupled onto solid support. Consequently, only glycosylation site information is provided while glycan structure is destroyed and lost. Despite this limitation, we believe that thiazolidine chemistry-based solid-phase extraction, as an alternative enrichment method, would expand the arsenal of MS analysis of N-glycoproteome from complex biological samples. Author contribution statement Thanks so much. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Please cite this article as: Y. Cai et al., A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of NGlycoproteome, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.001