English

• Protein glycosylation, as one of the most important and common post-translational modifications, is consanguineously relevant to multiple biological processes [1]. It is well known that glycoproteins serve as many disease markers and treatment labels in clinical diagnoses, but the low abundance and weak ionization efficiency of glycoproteins make the detection of glycoproteins challenging in mass spectrometry (MS) measurements [2, 3]. Until now, a wide variety of strategies for enriching glycopeptides have been studied including lectin affinity chromatography [4, 5], hydrazide chemistry [6, 7], boronic acid affinity chromatography [8-11], and hydrophilic interaction liquid chromatography (HILIC) [12-16]. Among them, HILIC method for glycopeptide enrichment based on hydrophilic partitioning and other interactions such as hydrogen bonding, electrostatic and dipole interactions, receives increasing attention because of its high sensitivity, good reproducibility, excellent MS compatibility, and easy operation [17, 18]. Accordingly, many kinds of hydrophilic materials have been designed and used for glycopeptide enrichment such as magnetic particles, graphene-based materials, and metal-organic frameworks [19-21]. Nevertheless, these materials still have difficulties in modifying high density of hydrophilic groups due to large steric hindrance, complicated synthesis steps, and harsh conditions [22, 23].

  Chitosan (CS), as a kind of natural polymer normally obtained by alkaline deacetylation of chitin, has become a very attractive biomaterial with non-toxicity, biocompatibility, as well as biodegradability [24, 25]. In addition, high density of amino and hydroxyl groups in CS molecule endow it with hydrophilicity and abundant reactive sites. CS has been used to design adsorbents toward a lot of substances, such as metal ions [26, 27], dyes [28, 29], environmental pollutants [30, 31], and proteins [32, 33]. Based on the hydrophilic interactions between plenty of hydrophilic groups of CS and glycopeptides, CS-coated magnetic colloidal nanocrystal cluster [34], highly cross-linked porous CS microsphere [35], and honeycomb CS membrane [23] were synthesized and used for glycopeptide enrichment. Unfortunately, it should be noted that the inherent inflexibility and poor acid resistance of CS are not conducive to the enrichment performance [36, 37]. As an alternative, the introduction of functional groups onto CS makes it possible to obtain improved enrichment capacity. For instance, poly(glycerol methacrylate) and iminodiacetic acid with hydrophilic groups were grafted onto the surface of CS to increase the hydrophilicity, thus enhancing the enrichment performance toward glycopeptides [38]. It can be deduced that the introduction of hydrophilic functional groups onto the surface of CS plays a positive role in improving the affinity between glycopeptides and CS-based HILIC materials, while the search of hydrophilic molecule that can be grafted onto CS is still on its way.

  Zwitterionic molecules containing both positive and negative groups exhibit attractive hydrophilicity [39]. L-Cysteine (L-Cys), as a zwitterionic hydrophilic compound, possesses polar groups including thiol, amino, and carboxyl groups, so L-Cys has shown great potential in the construction of hydrophilic materials for the efficient enrichment of glycopeptides [22, 40-42]. Besides, the affinity between thiol groups of L-Cys and metal nanoparticles allows L-Cys to be easily anchored to various types of substrates. Wu et al. [43] prepared L-Cys-modified silica microsphere columns for glycopeptide enrichment. In Ma et al.′s study [44], L-Cys-functionalized metal-organic framework with high hydrophilicity was applied to the capture of glycopeptides. There is no doubt that higher hydrophilicity will be obtained if L-Cys is anchored to a hydrophilic substrate. Motivated by the above information, a HILIC material designed by anchoring L-Cys onto the surface of hydrophilic CS can be anticipated to capture glycopeptides efficiently. To the best of our knowledge, no HILIC material combining the hydrophilicity of CS and L-Cys has been applied for glycopeptide enrichment.

  Herein, we developed magnetic L-Cys-functionalized CS (designed as Fe₃O₄@CS@Au-L-Cys) and applied to the specific capture of glycopeptides by utilizing the hydrophilicity of CS and L-Cys. The preparation of Fe₃O₄@CS@Au-L-Cys nanocomposites is illustrated in Scheme 1. CS was crosslinked with glutaraldehyde as the crosslinker and then coated onto the surface of Fe₃O₄ to obtain Fe₃O₄@CS. Au nanoparticles (Au NPs) were embedded onto the surface of Fe₃O₄@CS by electrostatic interaction to obtain Fe₃O₄@CS@Au, and then L-Cys molecules were grafted onto Fe₃O₄@CS@Au via Au-S bond between Au NPs and –SH of L-Cys to obtain Fe₃O₄@CS@Au-L-Cys. The synthesized Fe₃O₄@CS@Au-L-Cys possesses the following advantages. Firstly, abundant hydrophilic groups of CS provide not only attachment sites for Au NPs but also affinity sites toward glycopeptides. Secondly, the introduction of L-Cys with hydrophilic groups enhances hydrophilicity of this material, in other words, the hydrophilicity of both CS and L-Cys contributes to the efficient enrichment of glycopeptides. Thirdly, this material with superparamagnetism can be rapidly separated by an external magnet, which facilitates further MS analysis. Fe₃O₄@CS@Au-L-Cys nanocomposites exhibit high enrichment efficiency and selectivity for glycopeptide enrichment and were successfully applied to the enrichment of glycocpeptides from standard proteins digests and real complex bio-samples.

  Scheme 1

  

  Scheme 1.  (a) Synthesis process of Fe₃O₄@CS@Au-L-Cys and (b) typical enrichment process of glycopeptides by Fe₃O₄@CS@Au-L-Cys.

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  To confirm the successful synthesis of Fe₃O₄@CS@Au-L-Cys, FT-IR spectra of Fe₃O₄, Fe₃O₄@CS, and Fe₃O₄@CS@Au-L-Cys were determined. As indicated in Fig. 1a, the stretching vibration of Fe–O at 576 cm⁻¹ exists in the spectrum of unmodified Fe₃O₄. It can be seen that the peaks at 1061 cm⁻¹ (C–O–C stretching vibration in CS ring backbone), 1405 cm⁻¹ (–C–O stretching of primary alcoholic group in CS), 1627 cm⁻¹ (C=N stretching vibration formed by the reaction of CS and glutaraldehyde), and 2936 and 2867 cm⁻¹ (asymmetric and symmetric stretching vibration of C–H bond) exist in the spectrum of Fe₃O₄@CS. In addition, the peak at 1562 cm⁻¹ can be attributed to N–H bending vibration and the broad absorption band at around 3276 cm⁻¹ represents O–H stretching vibration [45, 46]. All the above evidences confirm that CS was successfully coated onto Fe₃O₄ surface, and both amino and hydroxyl groups are present on the surface of Fe₃O₄@CS. Au NPs formed by Na₃Cit as the reducing agent were embedded onto the surface of Fe₃O₄@CS, so the spectrum of Fe₃O₄@CS@Au shows peaks at 1713 and 1374 cm⁻¹ belonging to C=O and C–O stretching vibrations of carboxyl groups. In the spectrum of Fe₃O₄@CS@Au-L-Cys, the appearance of wide shape characteristic peak of −COOH at 3040 cm⁻¹ and the fingerprint peaks of L-Cys below 1000 cm⁻¹ (shaded area) clearly confirm L-Cys functionalization [44].

  Figure 1

  

  Figure 1.  (a) FT-IR spectra of Fe₃O₄, Fe₃O₄@CS, Fe₃O₄@CS@Au, and Fe₃O₄@CS@Au-L-Cys. (b) XPS pattern of Fe₃O₄@CS@Au-L-Cys. (c) VSM profiles of Fe₃O₄ and Fe₃O₄@CS@Au-L-Cys. (d) Water contact angles of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys.

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  The elemental composition of Fe₃O₄@CS@Au-L-Cys was tested by XPS determination. As shown in Fig. 1b, in addition to the characteristic peaks of Fe 2p, C 1s, N 1s, and O 1s, the signals of S 2p and Au 4f also appear, indicating the introduction of Au NPs and the grafting of L-Cys. Additionally, TEM images of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys (Fig. S1 in Supporting information) demonstrate the coating of CS onto Fe₃O₄ and the introduction of Au NPs with an average diameter of 40 nm. In summary, the above FT-IR, XPS, and TEM results clearly indicate the successful preparation of Fe₃O₄@CS@Au-L-Cys.

  XRD patterns of Fe₃O₄, Fe₃O₄@CS, Fe₃O₄@CS@Au, and Fe₃O₄@CS@Au-L-Cys were determined from 20° to 80° (2θ). As shown in Fig. S2a (Supporting information), six diffraction peaks at 2θ of 30.2°, 35.6°, 43.2°, 53.5°, 57.1°, and 63.0° exist in the curve of Fe₃O₄ NPs, which are consistent with those of standard Fe₃O₄ according to JCPDS No. 19–0629 [47, 48]. There are no other diffraction peaks in the spectrum of Fe₃O₄@CS, indicating that the coating of CS does not damage the crystal structure of Fe₃O₄. In the case of Fe₃O₄@CS@Au, there are not only the characteristic peaks of Fe₃O₄, but also four additional peaks of Au NPs at 2θ of 38.3°, 44.6°, 64.8°, and 77.7°, which can be attributed to (111), (200), (220), and (311) planes of Au NPs (JCPDS No. 04–0784) [17]. Such results prove that Au NPs were successfully loaded onto Fe₃O₄@CS. While for Fe₃O₄@CS@Au-L-Cys, the characteristic peaks of Fe₃O₄ and Au NPs also appear, indicating that the introduction of L-Cys does not affect the crystal structure of Fe₃O₄ and Au.

  The thermal stability of Fe₃O₄ and Fe₃O₄@CS@Au-L-Cys nanocomposites was characterized within the temperature range from 30 ℃ to 800 ℃. As shown in Fig. S2b (Supporting information), Fe₃O₄ NPs show excellent thermal stability and the weight loss is only 2.2%. However, the thermal stability of Fe₃O₄@CS@Au-L-Cys nanocomposites is not as good as that of Fe₃O₄ NPs. In the thermogram of Fe₃O₄@CS@Au-L-Cys nanocomposites, the sight weight loss (~10%) below 150 ℃ can be ascribed to the removal of water in the material. In the range from 150 ℃ to 460 ℃, there is an obvious wide range of weight loss (~47.15%), which might be caused by the rapid decomposition of CS and L-Cys. With further increase of temperature (> 460 ℃), the material exhibits slow weight loss (~11.16%), which can be attributed to the further degradation of the substance generated by CS degradation [49].

  The magnetic property of Fe₃O₄@CS@Au-L-Cys was investigated by using the magnetic hysteresis loop analysis. As shown in Fig. 1c, the maximum saturation magnetization value of Fe₃O₄@CS@Au-L-Cys is 27.9 emu/g. Compared to Fe₃O₄, the decrease in saturation susceptibility of Fe₃O₄@CS@Au-L-Cys is due to the introduction of the non-magnetic shell including CS and L-Cys. Nevertheless, Fe₃O₄@CS@Au-L-Cys can gather within 15 s using an external magnet, indicating that the material has superparamagnetic characteristics to guarantee its rapid magnetic separation from solution. Additionally, the stability of Fe₃O₄@CS@Au-L-Cys was evaluated (Fig. S3 in Supporting information). Before and after storage for one month, the solutions are both clear with the help of a magnet, indicating that Fe₃O₄@CS@Au-L-Cys is stable.

  To evaluate the hydrophilicity of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys, their water contact angel measurements were performed. As shown in Fig. 1d, the water contact angles of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys are 51.7° and 34.6°, demonstrating that both of them are typical hydrophilic materials. Obviously, the hydrophilicity of Fe₃O₄@CS@Au-L-Cys is higher than that of Fe₃O₄@CS, which further proves that the introduction of L-Cys contributes to the improvement of hydrophilicity. Fe₃O₄@CS@Au-L-Cys combining the hydrophilicity of CS and L-Cys has wide application prospects in the enrichment of hydrophilic glycopeptides.

  The enrichment performance of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys toward glycopeptides from HRP tryptic digests (1000 fmol/µL) was investigated (Fig. 2). As displayed in Fig. 2a, the signal intensity of glycopeptides was severely suppressed by MALDI-TOF MS direct analysis because of the presence of non-glycopeptides. However, after enrichment by Fe₃O₄@CS, the signals of glycopeptides were enhanced significantly and most of the interferences of non-glycopeptides were eliminated (Fig. 2b). This result implies that CS coated onto the surface of Fe₃O₄ NPs plays an essential role in the enrichment of glycopeptides owing to the hydrophilic groups in CS. Then, the enrichment of HRP tryptic digests with Fe₃O₄@CS@Au-L-Cys was performed and a total of 27 glycopeptides were identified in the MS spectrum (Fig. 2c). Detailed information of the enriched glycopeptides is listed in Table S1 (Supporting information). Obviously, based on the superior hydrophilicity of CS and L-Cys, more glycopeptides were identified with strong signals. As is expected, the introduction of hydrophilic L-Cys enhances the surface hydrophilicity and further promotes the affinity interaction with glycopeptides. Additionally, the enrichment of IgG tryptic digests by Fe₃O₄@CS@Au-L-Cys was evaluated (Fig. S4 in Supporting information). After enrichment by Fe₃O₄@CS@Au-L-Cys, 33 glycopeptides were clearly observed in the MS spectrum, indicating the unbiased enrichment ability of Fe₃O₄@CS@Au-L-Cys toward glycopeptides with different glycan types. Detail information of the enriched glycopeptides from IgG digests is listed in Table S2 (Supporting information).

  Figure 2

  

  Figure 2.  MALDI-TOF mass spectra of tryptic digests of 1000 fmol/µL standard HRP (a) before enrichment and after enrichment by (b) Fe₃O₄@CS and (c) Fe₃O₄@CS@Au-L-Cys. "*" indicates glycopeptide.

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  Considering the low abundance of glycopeptides and the interference of non-glycopeptides in real bio-samples, it is crucial to evaluate the detection sensitivity and selectivity of glycopeptides. The sensitivity of glycopeptides with the developed Fe₃O₄@CS@Au-L-Cys MS method was studied by the enrichment of different concentration of HRP tryptic digests (100, 10, 1 and 0.5 fmol/µL). When the limit of detection is as low as 0.5 fmol/µL, 11 glycopeptides were selectively captured according to S/N ratio greater than 3 (Fig. S5 in Supporting information), which further indicates that Fe₃O₄@CS@Au-L-Cys has a good enrichment performance toward glycopeptides even at low concentrations.

  The selectivity of glycopeptide enrichment by Fe₃O₄@CS@ Au-L-Cys was explored by adding BSA tryptic digests into HRP tryptic digests with different molar ratios. Few glycopeptides were detected without enrichment at HRP: BSA molar ratio is 1:100 (Fig. 3). After treated with Fe₃O₄@CS@Au-L-Cys, non-glycopeptides were effectively eliminated, while glycopeptides were clearly observed. It is noteworthy that glycopeptides can still be detected after enrichment with Fe₃O₄@CS@Au-L-Cys even when the molar ratio of HRP to BSA reaches 1:1000. The above results indicate that Fe₃O₄@CS@Au-L-Cys possesses great selectivity toward glycopeptides and can be expected to be applied as a highly efficient HILIC material for the capture of glycopeptides from real complex bio-samples.

  Figure 3

  

  Figure 3.  MALDI-TOF mass spectra of mixtures of HRP and BSA tryptic digests with a molar ratio of (a) 1:100 without enrichment and (b) 1:100, (c) 1:500, and (d) 1:1000 enriched by Fe₃O₄@CS@Au-L-Cys. "*" indicates glycopeptide.

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  For comparison, the detection sensitivity and selectivity of glycopeptides with Fe₃O₄@CS were also investigated (Figs. S6 and S7 in Supporting information). Obviously, the enrichment performance of Fe₃O₄@CS is inferior to that of Fe₃O₄@CS@Au-L-Cys for glycopeptides, proving that the introduction of hydrophilic L-Cys improves the glycopeptide enrichment performance. Besides, the comparison of the enrichment performance toward glycopeptides of Fe₃O₄@CS@Au-L-Cys with other HILIC materials is presented in Table S3 (Supporting information), which confirms the superior enrichment efficiency of Fe₃O₄@CS@Au-L-Cys.

  Considering the practical application, the reusability of Fe₃O₄@CS@Au-L-Cys was evaluated by the enrichment of glycopeptides in HRP tryptic digests in consecutive times. The same preliminary optimized loading (ACN/H₂O/TFA = 90/5/5) and elution (ACN/H₂O/TFA = 90/5/5) buffers were used during the whole enrichment and elution procedures. Results are shown in Fig. S8 (Supporting information), demonstrating that there is no significant difference between the signals of glycopeptides after the first and fifth cycle, confirming the excellent repeatability and stability of Fe₃O₄@CS@Au-L-Cys toward glycopeptide enrichment. In a word, the developed HILIC strategy possesses satisfactory sensitivity, selectivity, and repeatability, which makes it greatly potential to be applied to real complex bio-samples determinations.

  Encouraged by the above-obtained experimental results in the standard samples, Fe₃O₄@CS@Au-L-Cys was employed to enrich low-abundance glycopeptides from real complex bio-samples. Human serum, a commonly available clinical sample, was selected to evaluate the enrichment capacity of Fe₃O₄@CS@Au-L-Cys for glycopeptides. As shown in Figs. S9a and b (Supporting information), non-glycopeptides peaks dominate MS spectrum without enrichment, while 63 glycopeptides were identified from 2 µL normal human serum sample after Fe₃O₄@CS@Au-L-Cys enrichment. The detailed information is listed in Table S4 (Supporting information). To further evaluate the applicability of hydrophilic Fe₃O₄@CS@Au-L-Cys in complex bio-samples, human saliva was selected as another real complex bio-sample. Because the abundance of glycopeptides in human saliva is low, almost no corresponding glycopeptide peaks were found in the MS spectrum (Fig. S9c in Supporting information). Surprisingly, 37 glycopeptides were detected from 2 µL normal human saliva sample after enrichment with Fe₃O₄@CS@Au-L-Cys (Fig. S9d in Supporting information). Detailed information is listed in Table S5 (Supporting information). The comparison of the number of glycopeptides captured from real complex bio-samples of Fe₃O₄@CS@Au-L-Cys with the reported materials is shown in Table S6 (Supporting information). All the above results confirm that Fe₃O₄@CS@Au-L-Cys possessing high enrichment capacity toward glycopeptides has great application prospects in the analysis of real complex bio-samples.

  In summary, the main feature of this work is the development of a novel hydrophilic material named as Fe₃O₄@CS@Au-L-Cys for the efficient enrichment of glycopeptides. Uniting good hydrophilicity and biocompatibility of CS and L-Cys and superparamagnetism of Fe₃O₄, Fe₃O₄@CS@Au-L-Cys exhibited good enrichment performance toward glycopetides from tryptic digests of standard glycoproteins. The glorious enrichment performance of this hydrophilic material was largely determined by the great hydrophilicity of L-Cys immobilized onto hydrophilic magnetic CS substrate. Moreover, in practical application, Fe₃O₄@CS@Au-L-Cys exhibited great availability in capturing glycopeptides from real bio-samples like human serum and saliva. We expect that this material can provide inspiration for the development of hydrophilic materials and be extended to capture potential biomarkers in clinical research.

  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.

  Acknowledgments

  This work was financially supported by Open Project of State Key Laboratory of Supramolecular Structure and Materials, Jilin University, China (No. sklssm2022012) and the Fundamental Research Funds for the Central Universities, JLU, China.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.05.012.

  
  
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  Scheme 1  (a) Synthesis process of Fe₃O₄@CS@Au-L-Cys and (b) typical enrichment process of glycopeptides by Fe₃O₄@CS@Au-L-Cys.

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  Figure 1  (a) FT-IR spectra of Fe₃O₄, Fe₃O₄@CS, Fe₃O₄@CS@Au, and Fe₃O₄@CS@Au-L-Cys. (b) XPS pattern of Fe₃O₄@CS@Au-L-Cys. (c) VSM profiles of Fe₃O₄ and Fe₃O₄@CS@Au-L-Cys. (d) Water contact angles of Fe₃O₄@CS and Fe₃O₄@CS@Au-L-Cys.

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  Figure 2  MALDI-TOF mass spectra of tryptic digests of 1000 fmol/µL standard HRP (a) before enrichment and after enrichment by (b) Fe₃O₄@CS and (c) Fe₃O₄@CS@Au-L-Cys. "*" indicates glycopeptide.

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  Figure 3  MALDI-TOF mass spectra of mixtures of HRP and BSA tryptic digests with a molar ratio of (a) 1:100 without enrichment and (b) 1:100, (c) 1:500, and (d) 1:1000 enriched by Fe₃O₄@CS@Au-L-Cys. "*" indicates glycopeptide.

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