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Detecting Proteins Glycosylation by a Homogeneous Reaction System with Zwitterionic Gold Nanoclusters Jinan Li, Jing Liu, Zheyi Liu, Yuan Tan, Xiaoyan Liu, and Fangjun Wang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Analytical Chemistry

Detecting Proteins Glycosylation by a Homogeneous Reaction System with Zwitterionic Gold Nanoclusters Jinan Li,†,‡ Jing Liu,†,|| Zheyi Liu,†,‡, Yuan Tan,§,‡ Xiaoyan Liu,§ Fangjun Wang*,† †

CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China §

State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China ‡

University of Chinese Academy of Science, Beijing 100049, China

||

College of Pharmacy, Dalian Medical University, Dalian 116044, China.

ABSTRACT: Homogeneous gold nanoclusters (Au NCs) have been widely utilized in drug delivery, chemical sensing, bioassays and bio-labeling due to their unique physical and chemical properties. However, little attention has been paid to their application in detecting protein post-translational modifications. Herein, we describe development of a homogeneous reaction system with watersoluble zwitterionic Au NCs to capture glycopeptides from complex biological samples. The unique characteristics of Au NCs, such as their molecular-like properties, the excellent homogeneity in aqueous solution, the organic solvent responsive precipitation, and the easy preparation in only 4.5 hours, contribute to the high efficiency and high throughput for capturing the targeted glycopeptides. Compared with the conventional heterogeneous system with solid-state adsorbents, the number of characterized glycosylation sites was improved by 35%. Finally, an MS detection limit as low as 50 amol was achieved for the standard glycoprotein (IgG) and 1576 glycosylation sites from 713 glycoproteins were identified from only 60 µg of mouse liver protein. Data are available via ProteomeXchange with identifier PXD005635.

Monodisperse gold nanoclusters (Au NCs)1-4, linking between gold nanoparticles and single Au atoms, have been widely applied in drug delivery,5,6 chemical sensing,7,8 bioassays,9-11 and bio-labeling12 because of their unique physical and chemical characteristics. Au NCs and conventional Au nanoparticles (NPs) typically differ in terms of the moleculelike properties for the former, originating from strong quantum confinement and size-tunable transitions.3 Considering their high chemical stabilities and biocompatibilities, facile preparation and post-modification, homogeneity in aqueous solutions, and convenient separation by precipitation using organic solvents,1 water-soluble Au NCs are extremely suitable media for the in vivo and in vitro analyses of biological samples. However, few studies focus on the application of these Au NCs in analyzing protein post-translational modifications (PTMs). The synthesis of Au NCs involve protection by glutathione (GSH),13-15 phosphine,16,17 selenolate,18,19 and alkynyl ligand20. These protecting ligands play key roles in controlling the syntheses of Au NCs, as well as their photophysical, chemical, and biomedical properties. For e.g., thiol-protected Au NCs (Aux-RSy) are versatile gold nanoclusters because of their sizedependent, excited-state properties.21 Yu et al. have developed a versatile strategy for synthesizing thiol-protected Au NCs, with cysteine (Cys) as the protecting group.21 Meanwhile, Cys,

which is a common amino acid with high biocompatibility, serves as a promising zwitterionic monomer for the enrichment of glycopeptides via hydrophilic interaction chromatography (HILIC).22,23 Nevertheless, solid-liquid heterogeneous systems applied in conventional HILIC compromise the efficiency of glycopeptide capture because of the relatively high steric effect and limited interfacial mass transfer rates.24-26 On the other hand, liquid-phase homogeneous reaction systems play key roles in homogeneous catalysis and organic synthesis owing to advantages, e.g., no interface molecular transfer and high reaction rates.27-29 Unfortunately, marginal attention has been focused on the detection of target biological molecules using liquid-phase homogeneous reaction systems, which might easily eliminate the restricted mass transfer and steric hindrance.30-33 Recently, a pH-trigger soluble polymer with hydrazine group has been developed for glycopeptide capture.34 However, time-consuming polymerization is required to prepare this pH-responsive polymer, as well as a considerable amount (milligrams) of protein samples. In this study, we describe development of a homogeneous reaction system with water-soluble Au NCs for highly efficient detection of protein glycosylation for the first time. Briefly, Cys ligands were used as the protecting groups for Au NCs, and then the as-prepared Au NC aqueous solution was

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directly incubated with tryptic digests of protein samples, followed by the dropwise addition of acetonitrile (ACN). Au NCs were gradually precipitated from the solution, in addition to the increase in the ACN concentration. Meanwhile, the glycopeptides within the solution were co-precipitated with Au NCs because of the strong hydrophilic interaction between the glycan chain and zwitterionic Cys (Scheme 1).26,27 The homogeneous system with water-soluble zwitterionic Au NCs exhibited several advantages. First, Cys-protected Au NCs were rapidly synthesized (~4.5 h) by a convenient, economical route, and the Au NC aqueous solution was directly utilized to capture glycopeptides without any additional chemical modification. Furthermore, mass transfer between the incubated glycopeptides and the water-soluble Au NCs is not restricted, and the glycopeptides and Au NCs are gradually co-precipitated by the dropwise addition of ACN. Compared to conventional heterogeneous systems with solid-phase affinity adsorbents, homogenous systems with Au NCs can significantly improve the detection coverage and sensitivity of protein glycosylation. The synthesized zwitterionic Au NCs exhibited good monodispersity, with an average diameter of approximately 2.0 nm (Figure 1A). Furthermore, Au NCs exhibited a broad absorption band in the UV-vis spectrum (Figure 1B), and absorbance at ~520 nm, corresponding to the surface plasmon resonance band of conventional Au NPs, was not observed.35 Thus, zwitterionic Au NCs are synthesized in a facile manner, without the generation of large-sized Au NPs. The X-ray photoelectron spectroscopy (XPS) binding energies (BEs) of Au 4f5/2 and Au 4f7/2 were 87.98 and 84.28 eV for the Au NCs (Figure 1C), revealing that Au0 and Au+ co-exist in the Au NCs.36 In addition, the Au NCs exhibited high stability after storage at 4 °C in the dark over 2 months. Cys-protected Au NCs exhibited good solubility in aqueous solutions, and the Au NCs were collected by centrifugation after the addition of organic solvents or salt. As Cys has been reported to be a good zwitterionic monomer for hydrophilic interaction,22,23 Cys-protected Au NCs are speculated to be directly used for the interaction and capture of glycopeptides. Furthermore, the co-incubated glycopeptides could access the Au NCs with unrestricted mass transfer owing to the fact that the Au NC solution was homogeneous, which might be extremely beneficial in the efficient enrichment of the glycopeptides from complex biological matrixes. Hence, a homogeneous aqueous system with Cys-protected Au NCs is developed for the capture of glycopeptides, and glycopeptides gradually co-precipitate with Au NCs by the slow addition of ACN (Scheme S2 in Supporting Information). For comparison, a heterogeneous system with lyophilized solid-state Au NCs was developed for the enrichment of glycopeptides from the sample solution with 88% (v/v) ACN, and the procedures were the same as those described previously with other affinity adsorbents.30,31 First, the performance of the homogeneous system with Au NCs was evaluated for the enrichment of glycopeptides using two representative glycoproteins-horseradish peroxidase (HRP) and human serum immunoglobulin G (IgG), respective-

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ly, with different glycosylation degrees and molecular weights. Briefly, the tryptic peptide mixture was first incubated with the Au NC aqueous solution. Second, the ACN solution with 1% (v/v) trifluoroacetic acid (TFA) was added dropwise, leading to the simultaneous precipitation of Au NCs and glycopeptides. Next, the precipitated glycopeptides were collected by centrifugation and finally eluted into an aqueous solution with 30% (v/v) ACN and 0.1% (v/v) TFA after two washing steps (Figure S1 in the Supporting Information). If 0.5 pmol of the protein digests was directly analyzed by MALDI-TOF MS, only a few glycopeptides (H13, H18 and H23 for HRP, I7 and I12 for IgG) were observed in the mass spectra, with low intensity and low signal-to-noise ratios (SNRs), owing to the suppression of highly abundant nonglycosylated peptides in the mass spectra (Figures 2A a and B a).37,38 After the enrichment of the glycopeptides using the homogeneous system with Au NCs, 23 glycopeptides were clearly detected with improved signal-to-noise ratios (SNRs) for HRP tryptic digests; in particular, H7 exhibited a 23 times higher SNR (Figure 2A and Supporting Dataset 1-2). In addition, results were obtained for the analysis of glycopeptides for IgG tryptic digests (Figure 2B and Supporting Dataset 3-4). After the deglycosylation of the enriched glycopeptides by PNGase F, all of the signals corresponding to the typical glycopeptides from IgG disappeared, and signals corresponding to only two deglycosylated peptides (m/z = 1158 and 1190, respectively) were detected (Figure 2B). This result is consistent with those reported previously.30-32 In addition, this heterogeneous system with solid-phase Au NCs was applied to the enrichment of glycopeptides for comparison, and the intensities and SNRs of the detected glycopeptides were considerably less than those detected using the homogeneous aqueous system with water-soluble Au NCs (Figure S1 in the Supporting Information and Supporting Dataset 2-4). Therefore, the homogeneous system is more suitable for the enrichment of glycopeptides with considerably higher efficiencies. Different amounts of HRP tryptic digests were used to evaluate the glycopeptide detection limit of this homogeneous system with water-soluble Au NCs. As shown in Figure 3A, eight glycopeptides were clearly observed by MS for 50, 5, and 0.5 fmol HRP after treatment with the homogeneous Au NC system as described above (Figure 3A and Supporting Dataset 2). In addition, two glycopeptides (H2 and H7, respectively) were still detected with SNRs of 55 and 14, respectively, from only 50 amol of the HRP tryptic digests (Figure 3A and Supporting Dataset 2). In addition, the heterogeneous system with solid-state Au NCs was utilized, and glycopeptides were detected with considerably lower SNRs and intensities for 50, 5, and 0.5 fmol HRP digests, while glycopeptide signals were not observed for 50 amol HRP digests (Figure 3B). The glycopeptide detection limit observed for the homogenous system with Au NCs was considerably better than those observed for metal-organic frameworks, silica particles, and magnetic nanoparticles based on traditional heterogeneous systems with a detection limit of 0.1 fmol at best.30-32,39,40 Then, the glycopeptide enrichment recovery using the homo-

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geneous system with Au NCs was determined by a stable isotope labeling strategy as described in our previous study.30 The recovery yields for the two deglycosylated peptides with m/z of 1158 and 1190 were 80% and 91%, respectively. These values are comparable to those reported previously (Supporting Dataset 5).30-32 The homogeneous reaction system with Au NCs was utilized to detect the glycosylation of a mixture of mouse liver proteins extracted from 20 mice. In three experiments, 1357, 1337, and 1342 unique glycosylation sites corresponding to 633, 630, and 626 glycoproteins were easily detected, respectively. In total, 1576 glycosylation sites corresponding to 713 glycoproteins were detected from a 60 µg protein sample with a glycopeptide capture specificity of 45.8% (Supporting Dataset 6), and all glycosylation sites were in a consensus sequence of N-!P-S/T or rarely N-X-C motif (Figure S2 Supporting Information and Supporting Dataset 7). In contrast, only 1168 glycosylation sites corresponding to 583 glycoproteins were detected using the heterogeneous system with solidphase Au NCs under identical conditions by a lower capture specificity (20.0%, Supporting Dataset 8 and Figure S2 Supporting Information). The homogeneous system clearly exhibited considerably better performance for the enrichment of glycopeptides from a complex protein sample, and greater than 35% glycosylation sites were observed. In addition, greater than 85% of the glycosylation sites and proteins identified using the heterogeneous system were covered by the homogeneous strategy (Figure S3 Supporting Information). In conclusion, the potential of a monodisperse Au NC homogeneous reaction system for the analysis of protein PTMs was evaluated. To the best of our knowledge, this is the first time that Au NCs were feasibly introduced into the separated biological sample for comprehensive analyses of glycoproteins. A homogeneous reaction system, with gradual coprecipitation, was successfully developed for the highly efficient enrichment of glycopeptides. Compared to previous studies, such as those carried out with functionalized gold nanoparticles41,42 and functionalized magnetic nanoparticles,31,43 the incubated glycopeptides can access water-soluble Au NCs with unrestricted mass transfer, and the glycopeptides and Au NCs gradually co-precipitated with the dropwise addition of ACN. Compared to conventional heterogeneous systems with solid-phase affinity adsorbents, the homogenous system with Au NCs significantly improves the detection coverage and sensitivity of glycosylated protein. In addition, a better detection limit as low as 50 amol was achieved.34 Furthermore, the properties can be simply tuned by changing the protecting ligands. Considering these unique properties, Au NCs are expected to emerge as promising media for analyzing biological samples.

ASSOCIATED CONTENT

for capturing glycopeptides and mass spectrometry detection, and supplemental figures] (PDF) and supporting datasets (xlsx)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: +86-411-84379576, Fax:

+86-411-84379620

ACKNOWLEDGMENT Financial supports are gratefully acknowledged for the China State Key Basic Research Program Grant (2013CB911203), China State Key Research Grant (2016YFF0200504), the National Natural Science Foundation of China (21675152), and the Youth Innovation Promotion Association CAS (2014164) to F.W.

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Scheme 1

Illustration of the capture of Au NC-based glycopeptides with a homogeneous system.



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Figure 1


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(A) Typical TEM image of as-prepared Au NCs. Scale bar, 20 nm. (B) UV-vis absorption spectrum and (C) XPS spectrum of Au 4f for as-prepared Au NCs.



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Figure 2.

MALDI-TOF MS spectra of tryptic digests of (A) horseradish peroxidase (0.5 pmol), direct analysis (a) and after enrichment by Au NCs in a homogeneous system (b) and (B) human serum IgG (0.5 pmol), direct analysis (a), after enrichment by Au NCs in a homogeneous system (b) and the deglycosylation by PNGase F (c).


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Figure 3.



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MALDI-TOF MS spectra of the HRP tryptic digest of different concentrations after enrichment by the homogeneous system with soluble Au NCs (A) and the heterogeneous system with solid-phase Au NCs (B). 50 fmol (a), 5 fmol (b), 0.5 fmol (c) and 0.05 fmol (d).


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