Letter pubs.acs.org/ac
Highly Sensitive Method for Specific, Brief, and Economical Detection of Glycoproteins in Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis by the Synthesis of a New Hydrazide Derivative Weitao Cong,†,∥,¶ Ayi Zhou,†,¶ Zhiguo Liu,†,∥,¶ Jiayi Shen,† Xuan Zhou,§ Weijian Ye,† Zhongxin Zhu,†,∥ Xinliang Zhu,† Jianjun Lin,*,‡ and Litai Jin*,†,∥ †
School of Pharmacy, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P. R. China The Affiliated Xiangshan Hospital of Wenzhou Medical University, Ningbo, Zhejiang 41000, P. R. China § Ningbo First Hospital, Ningbo, Zhejiang 315000, P. R. China ∥ Wenzhou Undersun Biotechnology Co., Ltd., Wenzhou, Zhejiang 325000, P. R. China ‡
S Supporting Information *
ABSTRACT: A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively.
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high cost, which has greatly limited its wide application in most laboratories. In our previous study, we have developed two gel-separated glycoprotein staining methods based on dansylhydrazine12 and 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide,13 respectively. The detection limits of these two staining methods are both 4−8 ng, which is comparable with that of the Pro-Q Emerald 488 stain but lower than the Pro-Q Emerald 300 stain. Thus, in this study, a further step was taken toward a more simple, sensitive, economical, and MS-compatible staining method for gel-separated glycoproteins. A new hydrazide derivative (UGF202, in Scheme S1, Supporting Information) was designed and synthesized to label glycoproteins via the periodate/Schiff’s base (PAS) mechanism.14 Glycoproteins down to 0.5−1 ng could be selectively visualized by the newly developed UGF202 stain within 75 min, which is 2- and 4-fold higher than that of Pro-Q Emerald 300 and 488, respectively. Thus, this UGF202 stain is an advancement for gel-separated glycoprotein detection compared to the most commonly used Pro-Q Emerald staining kits.
lycosylation is the process by which a sugar is covalently attached to a target protein, which is one of the most common forms of post-translational modification of proteins. This modification serves various functions, for example, the degree and type of glycosylation depends on the status of cells and may be linked to certain diseases.1 Thus, discovery and identification of these modified peptides and proteins are important. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is currently one of the most reliable methods available for the separation, identification, and characterization of glycoproteins.2 There are many approaches employed for the visualization of glycoproteins in electrophoresis, including acid fuchsin,3 basic fuchsin,4 alcain blue,5,6 dansylhydrazine,7,8 and fluorescein-5-thiosemicarbazide.9 Recently, two fluorescence-based detection technologies, referred to as Pro-Q Emerald 30010 and 488 stains,11 commercially obtained from Invitrogen, have been applied for the direct detection of protein glycosylation in polyacrylamide gels, which provide down to 1−2 and 4−8 ng glycoprotein detection limits, respectively. Compared with the traditional approaches mentioned above, Pro-Q Emerald staining kits offer significant advantages, such as sensitivity, specificity, and compatibility with subsequent protein analysis. However, they both suffer from the major drawback of © 2015 American Chemical Society
Received: September 22, 2014 Accepted: January 7, 2015 Published: January 7, 2015 1462
DOI: 10.1021/ac503543z Anal. Chem. 2015, 87, 1462−1465
Letter
Analytical Chemistry
The synthetic route of UGF202 is shown in Scheme S1 (in Supporting Information), and the synthetic product was fully characterized by 1H NMR, 13C NMR, and ESI-MS (in Supporting Information). UGF202, a new hydrazide derivative, had an excitation peak of 402 nm and an emission peak of 475 nm (Figure S1, Supporting Information). After electrophoresis, gels were fixed in 50% methanol (MeOH) and 5% acetic acid (HAc) for 30 min. The gels were subsequently oxidized with 0.5% periodic acid (HIO4) and 3% HAc for 20 min and immersed in 0.8% ascorbic acid solution for 5 min to neutralize the excess HIO4 in the gel matrix. Finally, the gels were incubated in 0.001% UGF202, 40% ethanol (EtOH), and 5% N,N-dimethylformamide (DMF) staining solution for 20 min to specifically detect glycoproteins in SDS-PAGE. It should be noted that, in every step, a volume equivalent to ∼10-fold excess of the gel volume should be used, and all the steps were carried out at room temperature with gentle shaking. The sensitivity and specificity of the UGF202 stain was compared with Pro-Q Emerald staining kits, and 1-D SDS-PAGE gel images of glycoprotein markers obtained by the different stains were compared as shown in Figure 1. SYPRO Ruby and Pro-Q Emerald 300 and 488 were essentially performed according to the manufacturer’s instructions from Invitrogen. The results showed that the detection limit of the UGF202 stain was 0.5−1 ng for transferrin and α1-acid glycoprotein (analyzed by Image Lab 2010 software). Correspondingly, for Pro-Q Emerald 300 and 488 stained gels, the detection limits were 1−2 and 4−8 ng, respectively, which demonstrated that the UGF202 stain had a higher sensitivity compared to the Pro-Q Emerald staining kits. Furthermore, to verify the efficiency of the UGF202 stain, total proteins extracted from human serum, mouse liver, human fibroblasts, and mouse heart as real biosamples were separated in 1-D SDS-PAGE and detected by the different methods. According to the results (Figure 2), we could find that the UGF202 stain provided similar or better sensitivity and specificity for the detection of glycoproteins compared to the Pro-Q Emerald staining kits. Of particular interest to glycoproteomic endeavors is the application of the UGF202 stain to provide a sensitive and
Figure 1. Comparison of the sensitivity of the UGF202 staining method with Pro-Q Emerald staining kits in 1-D SDS-PAGE. Here, 2-fold serial dilutions of molecular weight standards ranging from 250 ng to 0.5 ng per single band are shown. (A) UGF202 stain; (B) Pro-Q Emerald 300 stain; (C) Pro-Q Emerald 488 stain; (D) SYPRO Ruby stain.
Figure 2. Comparison of the sensitivity of the UGF202 stain with Pro-Q Emerald staining kits in 1-D SDS-PAGE with total proteins from human serum, mouse liver, human fibroblasts, and mouse heart. Here, 2-fold serial dilutions of these total proteins (lanes 1−10, from left to right) were loaded into the gels. (A) UGF202 stain; (B) Pro-Q Emerald 300 stain; (C) Pro-Q Emerald 488 stain; (D) SYPRO Ruby stain. 1463
DOI: 10.1021/ac503543z Anal. Chem. 2015, 87, 1462−1465
Letter
Analytical Chemistry specific method for visualization of 2-D SDS-PAGE. Thus, total proteins extracted from human serum were separated and detected by the UGF202 stain and Pro-Q Emerald staining kits (Figure 3). The results showed that most protein spots detected
Figure 4. Comparison of specificity of glycoprotein detection by the UGF202 stain with Pro-Q Emerald staining kits with different protein samples. (+): PNGase F deglycosylation treated; (−): nontreated. (A) Transferrin; (B) human serum total proteins; (C) mouse liver total proteins.
total proteins. According to the results (Figure S2, Supporting Information), the UGF202 stain provided similar specificity and better sensitivity compared to that of the Pro-Q Emerald staining kits. In addition to sensitivity and specificity, gel image reproducibility is another crucial factor for glycoprotein staining methods. To validate reproducibility, three UGF202 and Pro-Q Emerald 300 stained 2-D gels were analyzed by ImageMaster 2D Platinum v7.0, respectively. The intensity of each spot was quantified by calculation of spot volume after normalization of the image using a total spot volume normalization method multiplied by the total area of all the spots. As shown in Figure S3, Supporting Information, the average correlation for the UGF202 stain was 0.889, while the value for Pro-Q Emerald 300 was 0.876, which indicated that the reproducibility of these two staining methods was approximately the same. Moreover, the linearity correlation between glycoprotein amounts and their fluorescence intensity was examined. Three marker glycoproteins were applied to SDS-PAGE, from 0.5 to 250 ng, in a 2-fold dilution for the UGF202 and Pro-Q Emerald 300 stains (Figure S4, Supporting Information). In this range, the correlation coefficient of UGF202 was from 0.982 to 0.994, which was comparable with that of the Pro-Q Emerald 300 stain. In order to determine the compatibility of the UGF202 stain with LC-MS/MS, 19 protein spots were selected and excised from 3 UGF202 stained human serum 2-D gels (as indicated in Figure 3), then digested with trypsin, deglycosylated with PNGase F, and finally identified by LC-MS/MS. According to the results (shown in Table S1, Supporting Information), all the selected spots were identified as glycoproteins. In total, 59 Nlinked glycosylation sites were verified in the 7 identified glycoproteins. In addition, we also investigated the spectrum for two representative deglycosylated peptides K.HSTIFEN*LANK.A (spot 2 in Figure 3) and K.YLGN*ATAIFFLPDEGK.L (spot 11 in Figure 3), and their N-glycosylation sites were assigned as N231 and N271, respectively (Figure 5). Finally, Table 1 compares and summarizes the characteristics of the UGF202 stain and Pro-Q Emerald 300 and 488 stains.
Figure 3. Comparison of 2-D gel images of proteins from human serum detected by the UGF202 stain and Pro-Q Emerald staining kits. The proteins were resolved in 3−10 linear pH gradient (IPG strips; 130 × 3 × 0.5 mm) and 11.5% SDS-PAGE (150 × 150 × 1 mm). Nineteen protein spots were excised from the UGF202 stained gels, digested with trypsin, deglycosylated with PNGase F, and then identified by LC-MS/MS.
by Pro-Q Emerald 300 and 488 stains could be visualized by the UGF202 stain. In addition, ImageMaster 2D Platinum v7.0 software was introduced to analyze 2-D gel-separated protein spots. The results showed that 186 spots were visualized by the UGF202 stain, while for Pro-Q Emerald 300 and 488 stains, the number of protein spots were 163 and 133, respectively. Furthermore, 151 spots in Pro-Q Emerald 300 stained gels and 121 spots in Pro-Q Emerald 488 stained gels could also be visualized in UGF202 stained gels. The results further indicated that the UGF202 stain provided similar or better sensitivity compared to Pro-Q Emerald staining kits for 2-D SDS-PAGE. To explore the specificity of glycoprotein detection in this novel UGF202 stain, the staining patterns of the glycoprotein marker (transferrin) and total proteins from human serum and mouse livers were compared with deglycosylated ones. PNGase F, an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hydrid, and complex oligosaccharides from N-linked glycoproteins, was used to remove glycosylation groups from these samples. After PNGase F treatment, all the samples for the UGF202 stained gels showed a characteristic decreased band signal and downward band shift, which were also observed for Pro-Q Emerald 300 and 488 stains (Figure 4). This suggested that the UGF202 stain showed similar specificity when compared with the Pro-Q Emerald staining kits. In addition, to further evaluate UGF202 stain specificity, a glycoprotein enrichment kit was obtained from Thermo Scientific and used to extract glycoproteins from human serum 1464
DOI: 10.1021/ac503543z Anal. Chem. 2015, 87, 1462−1465
Letter
Analytical Chemistry
Figure 5. Glycosylation site localization of glycosylated peptides. (A) K.HSTIFEN*LANK.A (spot 2 in Figure 3); (B) K.YLGN*ATAIFFLPDEGK.L (spot 11 in Figure 3).
2013R10020); Public Projects of Zhejiang Province (2013C37050); the Science and Technology Program of Ningbo (2013C50040); the Natural Science Foundation of Ningbo (2013A6110165); the Science and Technology Program of Longwan (2013YS01).
Table 1. Comparison of the Characteristics of UGF202 Stain with Pro-Q Emerald Staining Kits for the Glycoprotein Visualization in SDS-PAGE methods
UGF202
Pro-Q Emerald 300
Pro-Q Emerald 488
elapsed time sensitivity cost (per use) steps
∼75 min 0.5−1 ng $0.8 4
overnight 1−2 ng $70 11
overnight 4−8 ng $50 12
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The results showed that, compared with Pro-Q Emerald 300 and 488 stains, the UGF202 stain is relatively simple (4 steps), timesaving (75 min), and low cost ($ 0.8 per use) and provides better sensitivity (0.5−1 ng) for glycoprotein detection. Considering the characteristics of this novel staining method, it is concluded that the UGF202 stain is a simple, specific, sensitive, and economical glycoprotein detection method compared with those of Pro-Q Emerald staining kits.
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ASSOCIATED CONTENT
* Supporting Information S
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: +86-577-86699790. E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions ¶
W.C., A.Z., and Z.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Key Scientific Instrument Development Program of China (2011YQ030139); the National Nature Science Foundation of China (31300657, 21472142); the Natural Science Foundation of Zhejiang Province (LY13C050002); Qianjiang talents Program (QJD1202019, 1465
DOI: 10.1021/ac503543z Anal. Chem. 2015, 87, 1462−1465
