Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility. - PDF Download Free (2024)

3512 Zhongxin Zhu1,2 ∗ Xuan Zhou1,3 ∗ Yang Wang1 ∗ Qing Yu1 Xinliang Zhu1 Chao Niu1 Weitao Cong1,2 ∗∗ Litai Jin1,2 1 School

of Pharmaceutical Sciences, Key Laboratory of Biotechnology Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang, P. R. China 2 Wenzhou Undersun Biotechnology Co. Ltd, Wenzhou, Zhejiang, P. R. China 3 Ningbo First Hospital, Ningbo, Zhejiang, P. R. China

Received March 20, 2014 Revised August 25, 2014 Accepted August 29, 2014

Electrophoresis 2014, 35, 3512–3517

Short Communication

Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2carboxylic acid hydrazide with weak influence on protein mobility A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4–8 ng glycoproteins (transferrin, ␣1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins. Keywords: 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide / Glycoprotein detection / Prestain DOI 10.1002/elps.201400351

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Glycosylation is a common posttranslational and cotranslational modification of extracellular and integral membrane proteins as well as certain intracellular proteins of eukaryotes [1, 2]. Most eukaryotic proteins are posttranslationally glycosylated at Asn residues with N-linked glycans, at Ser/Thr residues with O-linked glycans, or at the protein Cterminal of carboxylic acid with glycosyl phosphatidylinositol anchors [3, 4]. These glycosylations can alter a protein’s physical properties such as steric hindrance, charged state, or hydrophobicity, which contribute to the activity, recognition, immunogenicity, solubility, and stability of proteins [5]. Therefore, many physiological processes such as control of cell growth, cell migration, cell adhesiveness, tissue differentiation, and inflammatory reactions are closely associated with protein glycosylation [6, 7]. Currently, SDS-PAGE is the most commonly used technique for the separation, purification, identification, and quantification of glycoproteins [8]. There are two principal

Correspondence: Professor Litai Jin, Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical University, Wenzhou 325035, P. R. China E-mail: [emailprotected] Fax: +86-577-86699790

Abbreviations: BH, 4H-[1]-Benzopyrano[4,3-b]thiophene-2carboxylic acid hydrazide; DW, deionized water; FTSC, fluorescein 5-thiosemicarbazide; HIO4, periodic acid; MeOH, methanol C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

strategies that always applied for staining glycoproteins in PAGE: poststain and prestain. These two general approaches involve an overall mechanism where the probe dye is covalently conjugated to glycoproteins through a reaction between hydrazide and aldehyde generated by the oxidation of carbohydrade groups in glycoproteins. For the first method, poststain is a well-established technique that requires several additional steps to visualize gel-separated glycoproteins after electrophoresis. Dyes such as acid fuchsin [9], basic fushisn [10], alcain blue [11, 12], dansylhydrazine [13, 14], Pro-Q Emerald 488 [15], and Pro-Q Emerald 300 [16] have been successfully applied to detect glycoproteins by poststain. Among these methods, currently, gel-separated glycoproteins are most often detected by Pro-Q Emerald 300 and Pro-Q Emerald 488 stains, commercially obtained from InvitrogenTM . Pro-Q Emerald stains are breakthrough techniques that provide sensitive protocols for selective staining of gel-separated glycoproteins [15, 16]. Although the sensitivity and specificity are definite positives, the high costs of Pro-Q Emerald dyes have limited its application to high-throughput glycoproteomics in most laboratories. Additionally, Pro-Q Emerald dyes also include numerous time-dependent manual steps and involves the repeated changing of solutions. ∗

These authors have equally contributed to this work. Additional corresponding author: Dr. Weitao Cong, E-mail: [emailprotected]

∗∗

Colour Online: See the article online to view Fig. 4 in colour. www.electrophoresis-journal.com

Electrophoresis 2014, 35, 3512–3517

Meanwhile, in the prestaining method, glycoproteins are labeled prior to electrophoresis and then directly loaded onto the gel where separation takes place. No further steps are required, and the gels can be analyzed instantaneously after electrophoresis. However, prestaining methods are always challenged by the problem of electrophoretic mobility and band broadening [17,18]. To our knowledge, only a few probes have been applied to glycoprotein prestain, for example: fluorescein 5-thiosemicarbazide (FTSC) [19]. Therefore, it is still necessary to have a means for quick, sensitive, specific, and economical staining of gelseparated glycoproteins. According to our previous study, 4H[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) has been used for glycoprotein poststain in SDS-PAGE with high specificity and low cost. However, the operation was relatively time-consuming and was a complicated operation with numerous changes in solutions [20]. As another new attempt to seek better technology, BH prestain was devised in this study. For BH prestain, we noted that the specific capture of glycoproteins is also based on the oxidative reaction of hydroxyl groups on adjacent carbon atoms of carbohydrates to aldehydes by sodium periodate as mentioned above. As low as 4–8 ng glycoproteins could be specifically visualized immediately after electrophoresis by BH prestain, which is about 16-fold higher than that of FTSC prestain, and comparable with Pro-Q Emerald 488 stain that detected 4–8 ng of the same marker proteins. In this study, acrylamide, bis, ammonium persulfate, Tris base, glycine, SDS, iodoacetamide, glycerol, bromophenol blue, ammonium bicarbonate, trypsin, BH, FTSC, SYPRO Ruby, transferrin (glycoprotein, human, 80 kDa), BSA (bovine, 66 kDa), IgG (glycoprotein, rabbit, 50 kDa), ovalbumin (glycoprotein, chicken, 45 kDa), ␣1-acid glycoprotein (glycoprotein, bovine, 41 kDa), ␣-casein (phosphoprotein, bovine, 25 kDa), ␤-casein (phosphoprotein, bovine, 24 kDa), and avidin (glycoprotein, egg, 16 kDa) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). CHAPS, DTT, PMSF, urea, IPG strip, cover oil, and IPG buffer were from Amersham Biosciences (Uppsala, Sweden). PNGase F (Cat # P0704L) was purchased from New England BioLabs (Beverly, MA, USA). Pro-Q Emerald 488 glycoprotein staining kit (Cat # P21875) was from Invitrogen (Carlsbad, USA). Glycoprotein Affinity Isolation Kit was purchased from Thermo ScientificTM (Thermo Fisher Scientific, Bremen, Germany). All other chemicals used were of analytical grade and were obtained from various commercial sources. For 1D SDS-PAGE, before being applied onto the gels, molecular weight marker proteins or human serum total proteins (110 ␮g/␮L in deionized water (DW)) were mixed with equal volumes of 0.03 M periodic acid (HIO4 ) and incubated for 10 min at room temperature in the dark. Then, to remove residual HIO4 in the mixture, a user-friendly reducing substance, ascorbic acid (0.25 M, one fourth to equal volumes) was added into the mixture and incubated for 1 min at room temperature. After that, the protein mixtures were labeled with one fifth to equal volumes of 1% BH stock solution (dissolved in DMSO) for 30 min at 37°C in the dark. Finally, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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the BH-labeled protein solutions were diluted with 3X SDS sample buffer (0.15 M Tris-HCl (pH 6.8), 0.3 M DTT, 6% SDS, 30% glycerol, 0.3% bromophenol blue) for electrophoresis. Twofold serial dilutions of marker proteins and human serum total proteins were loaded onto the gel lanes, respectively. Electrophoresis was carried out on polyacrylamide slab gels (60 × 80 × 0.75 mm), using the discontinuous buffer system of Laemmli [21]. The 4.5% stacking gel was overlaid on the separation gel of 10% polyacrylamide with an acrylamide: Bis ratio of 30:0.8. The running buffer consisted of 0.025 M Tris, 0.2 M glycine, and 0.1% SDS. The gels were run in a Mini-protein III dual slab cell (Bio-Rad, Hercules, CA, USA) at a constant current of 22 mA per slab gel using a Power PAC 300 (Bio-Rad). For 2D SDS-PAGE, a desalted sample was suggested for IEF. Thus, the protein samples must be processed prior to IEF. For a 150 ␮L BH-labeled sample (150300 ␮g of proteins), firstly 600 ␮L methanol (MeOH) was added and mixed well by vortexing, then 150 ␮L chloroform was added and mixed well followed by addition of 450 ␮L DW. After that, the protein mixtures were centrifuged at 10 000 × g (4°C) for 5 min and the upper phase was discarded, keeping the white precipitation disc that forms between the upper and lower phases. Then, 450 ␮L MeOH was added and centrifuged at 10 000 × g (4°C) for 5 min. Supernatant was discarded and the pellet was dried in a vacuum centrifuge for 3 min, and then the pellet was resuspended in a rehydration buffer (8 M urea, 2% CHAPS, 2% IPG buffer, 0.04 M DTT, 1X nuclease solution, and a few grains of bromophenol blue) and then centrifuged at 15 000 × g for 15 min at 4°C to collect the protein-containing supernatant for subsequent application to 13 cm, IPG strips with pH gradient 3–10 (GE Healthcare). After overnight incubation at room temperature, the strips were subjected to first-dimension IEF using an Ettan IPGphor 3 system (GE Healthcare). Upon completion of the first-dimensional electrophoresis, IPG strips were first equilibrated for 15 min in an aqueous solution containing 50 mM Tris, 6 M urea, 30% glycerol, 2% SDS, and 55 mM DTT, pH 8.8, and then in a solution of the same composition, but containing 110 mM iodoacetamide instead of DTT. SDS-PAGE was performed at 20 mA per gel for 30 min and then 30 mA per gel until the bromphenol blue dye front had migrated off the far end of the gels. The images of BH prestain, Pro-Q Emerald 488 and SYPRO Ruby stains were generated using a Typhoon 9400TM scanner (Amersham Biosciences) with the resolution at 200 dpi. A 580-nm longpass emission filter, a 488-nm laser excitation source, and 500 V PMT were used for BH prestain and Pro-Q Emerald 488 stain, and a 610-nm longpass emission filter, a 532-nm laser excitation source, and 500 V PMT were used for SYPRO Ruby stain. Visualization of silver and CBBR stained gels were performed using a white-light scanner (UMAX PowerLook 2100XL, Umax Systems GmbH, Germany) connected to a computer. The images were exported in TIF format. Firstly, to explore the characteristics of the newly developed staining method, electrophoretic glycoproteins www.electrophoresis-journal.com

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Figure 1. Comparison of the sensitivity and specificity of different detection methods in 1D SDS-PAGE with standard marker proteins (A, B, C, D) and human serum total proteins (E, F, G, H). (A, E) BH prestain; (B, F) FTSC prestian; (C, G) Pro-Q Emerald 488 stain; (D, H) SYPRO Ruby stain. The amounts of standard marker proteins in each lanes are as follows: lane 1, 1000 ng/band; lane 2, 500 ng/band; lane 3, 250 ng/band; lane 4, 125 ng/band; lane 5, 64 ng/band; lane 6, 32 ng/band; lane 7, 16 ng/band; lane 8, 8 ng/band; lane 9, 4 ng/band; lane 10, 2 ng/band. For human serum total proteins, lanes 1–10 (from left to right) were twofold serial dilutions.

prestained with BH were compared with FTSC prestain and Pro-Q Emerald 488 stain in 1D SDS-PAGE (Fig. 1 and Table 1). FTSC prestain and SYPRO Ruby stain were essentially performed according to Gao et al. [19] and Berggren et al. [22], respectively, while Pro-Q Emerald 488 stain was essentially performed according to the instructions from InvitrogenTM . According to the results, we could clearly see that down to 4–8 ng of glycoproteins (transferrin, ␣1-acid glycoprotein) could be specifically detected by BH prestain, which is approximately 16-fold higher than that of FTSC prestain, and similar to that of Pro-Q Emerald 488 stain (Fig. 1A–C). Meanwhile, although an equal amount of each standard marker glycoprotein was prepared and loaded onto each lane, the different intensities of five glycoproteins provided

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

by BH prestain were primary contributed by the different glycosylation levels of these proteins. Furthermore, in order to confirm the sensitivity and compare the staining pattern, human serum total proteins acting as real biosamples were separated by 1DE and stained by different methods (Fig. 1E–G). The results demonstrated that the sensitivity and selectivity of BH prestain is much higher than FTSC prestain, and comparable with that of Pro-Q Emerald 488 stain. Additionally, among the current strategies for proteome study, 2DE is one of the most powerful tools for separating glycoproteins from a complex mixture. Therefore, we have applied the BH prestaining procedure to analyze 2D separated human serum total proteins (Fig. 2). The results showed that most of the spots detected by Pro-Q Emerald 488 stain could be visualized by

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Figure 2. Comparison of the sensitivity and specificity of different detection methods in 2D SDS-PAGE with human serum total proteins. (A) BH prestain; (B) Pro-Q Emerald 488 stain; (C) SYPRO Ruby stain.

Table 1. Comparison of the detection limit of BH prestain with Pro-Q Emerald 488 stain

Glycoprotein name

Transferrin IgG Ovalbumin ␣1-Acid glycoprotein Avidin

Staining method (ng/band) BH prestain

Pro-Q Emerald 488 stain

4–8 16–32 16–32 8–16 4–8

8–16 32–64 64–125 8–16 8–16

the BH prestain, which indicated that the sensitivity of the BH prestain is similar to that of Pro-Q Emerald 488 stain. Moreover, to determine whether covalent linkage of BH dye to marker proteins significantly alters their relative electrophoretic mobility, a comparison study between the BH labeled and unlabeled proteins was performed. As shown in Fig. 3, the migration rates of BH prestained marker protein bands are nearly identical to those of poststained protein bands, which might be attributed to the relative small molecular weight of the BH dye. To note, the bonding of BH with marker proteins is likely to broaden some protein bands to some extent, such as the diffusion of the ␣1-acid glycoprotein band in Fig. 3. Also, the compatibility property of BH with silver, CBBR and SYPRO Ruby were investigated by comparison of the characteristics of differently stained gels. The results showed that BH prestain was compatible with silver, CBBR and SYPRO Ruby stains, similar to that of Pro-Q Emerald 488 stain (Supporting Information Fig. 1). Silver stain and CBBR stain were essentially performed according to Jin et al. [23] and Choi et al. [24], respectively. Specificity is one of the most important indices for evaluating the performance of glycoprotein staining methods. In order to investigate the specificity of BH prestain, deglycosylation was utilized for human serum total proteins. The C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 3. Monitoring of the mobility of BH prestain. A 500 ng/band of standard marker proteins prestained (A) or not (B) with BH dye were separated by SDS-PAGE and restained by SYPRO Ruby.

removal of glycosylation groups from this sample was conducted with PNGase F. As shown in Supporting Information Fig. 2, in BH prestained gels, deglycosylation-treated human serum total proteins showed an extremely decreased band signal and band shifting, which was similarly observed in Pro-Q Emerald 488 stained gels. To our knowledge, this is a kind of deglycosylated form, caused by incomplete removal of deglycosylation groups on glycoproteins with PNGase F due to its substrate specificity. The results indicated that the specificity of BH prestain is comparable with Pro-Q Emerald 488 stain.

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Figure 4. MS/MS spectrum of the deglycosylated peptide K.DSGFQMNQLR.G from band 6 and K.AHYGGFTVQNEANKYQISVNK.Y from band 8 in Fig. 1. N represents the Asp residue formed via PNGase F deglycosylation from the glycosylation site at the Asn residue.

Continuing, lectin affinity enrichment is a widely used technique to isolate multiple types of glycoproteins from complex biological samples [25]. In this study, to further confirm BH prestain specificity, a ConA lectin Glycoprotein Isolation Kit from Thermo ScientificTM was introduced to enrich glycoproteins from human serum total proteins. As shown in Supporting Information Fig. 3, the sensitivity and specificity of BH prestain is comparable to that of Pro-Q Emerald 488 stain for glycoproteins analysis. In order to determine the MS compatibility of BH prestain, nine human serum glycoprotein bands (indicated in Fig. 1E) were excised from three replicated BH prestained gels and identified by MS following trypsin digestion and PNGase F deglycosylation (Supporting Information Table 1). The results suggested that BH prestain did not interfere with the in-gel trypsin digestion of BH-labeled glycoprotein and downstream protein identification. In total, 36 N-linked glycosylation sites were verified in the nine identified glycoproteins. In addition, we also investigated the spectrum for two representative deglycosylated peptides K.DSGFQMN*QLR.G (band 6 in Fig. 1) and K.AHYGGFTVQNEAN*KYQISVNK.Y (band 8 in Fig. 1), and their N-glycosylation sites were assigned as N129 and N366 , respectively (Fig. 4). Finally, the costs of BH and Pro-Q Emerald 488 stains were evaluated. For single use, BH prestain had a cost of $0.4 compared with the $50 needed for Pro-Q Emerald 488 stain (mini gel). In summary, BH prestain can provide favorable detection of glycoproteins with high selectivity and weak influence on protein mobility. Considering its quick, sensitive, specific, and economical characteristics, BH prestain might be a plausible alternative to the conventional Pro-Q Emerald 488 stain.

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

This work was supported by the National Key Scientific Instrument Development Program of China (2011YQ030139); the National Nature Science Foundation of China (31300657 and 81201245); the Zhejiang Provincial Natural Science Foundation (LY13C050002); the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents and the Science; Qianjiang talents Program (QJD1202019); Public Projects of Zhejiang Province (2013C37050); and the Science and Technology Program of Longwan (2013YS01). The authors have declared no conflict of interest.

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