Sensitive Western-Blot Analysis of Azide-Tagged Protein Post

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Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications (PTMs) Using Thermo-responsive Polymer Self-assembly Tong Liu, Wanjun Zhang, Zheng Zhang, Ming-Li Chen, Jian-Hua Wang, Xiaohong Qian, and Weijie Qin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04531 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Analytical Chemistry

Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications (PTMs) Using Thermo-responsive Polymer Self-assembly

Tong Liu,†, ‡ Wanjun Zhang,‡ Zheng Zhang,‡ Mingli Chen*, Xiaohong Qian‡ and Weijie Qin*, ‡

†

Jianhua Wang,†

†

Research Center for Analytical Sciences, College of Sciences, Northeastern University, Shenyang 110819, PR China ‡ State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, PR China

*Corresponding Author E-mail: [email protected] (Weijie Qin) and [email protected] (Mingli Chen)

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of

protein post-translational modifications (PTMs) is

hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermo-responsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermo-responsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still have important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

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Introduction As a powerful analytical technique for protein identification in complex biological samples, western-blot (WB) is easy to use, semi-quantitative and inexpensive, which makes it widely adopted in almost every biological laboratory for decades1. In this technique, target proteins separated by SDS-PAGE and transferred to PVDF membrane are identified by antibodies that recognize the target proteins and are visually detected via chemiluminiscent signal generated by the antibody conjugated enzyme, such as horseradish peroxidase (HRP)2. Detection specificity and sensitivity of western-blot (WB) largely relies on the quality of the antibodies and performance of the conjugated HRP, which is crucial for confidently distinguishing the true signal from background noise and for obtaining reliable and accurate results, especially for investigating low abundant proteins. False positive detection resulted from nonspecific binding of antibody to unrelated proteins is a common issue, due to the limited availability of high quality antibodies, especially in the analysis of protein post-translational modifications (PTMs). PTMs refer to the dynamic covalent modifications attached on proteins during or after protein biosynthesis. PTMs play many critical roles in various of cellular processes and regulate almost every aspect of proteins, such conformation, activity, cellular localization and protein-protein interaction3-5. PTMs analysis provides invaluable insights for physiological and pathological studies, but remains technically challenging using WB based technique. It is difficult for the antibodies to recognize and differentiate tiny PTMs, such as phosphorylation or O-GlcNAcylation attached on relatively huge proteins without 3



bias towards particular sequence motif of the substrate proteins6,7. Furthermore, for PTMs such as glycosylation or arginine/histidine phosphorylation, which are either poorly immunogenic or highly unstable, generating antibody itself becomes a formidable task8-11. Another major obstacle in WB analysis is the limited signal production by the traditional antibody conjugated HRP. The sensitivity of this colorimetric assay is hardly comparable with that of plasmon based tests12. Various methods have been proposed to amplify the chemiluminiscent signal of WB by attaching multiple antibody conjugated HRPs on certain carrier materials. For example, nanoparticles13, graphene oxide14 and dentrimer15 were reported as new carriers for the immobilization of multiple antibody conjugated HRPs with improved detection sensitivity in WB analysis. However, the binding between the immobilized antibodies on the carried materials and the target proteins on the PVDF membrane is challenging. The heterogeneous reaction between the immobilized antibodies on the solid/insoluble carrier materials and the membrane attached target proteins leads to significant interfacial mass transfer resistance, nonlinear reaction kinetics and high steric hindrance. These obstacles limit further improvement of the detection sensitivity of WB analysis. Therefore, new target proteins recognition mechanisms and signal amplification methods for WB analysis are in urgent needs.

To solve this problem, we synthesized triarylphosphine and HRPs functionalized environmental-responsive and self-assembling polymers for sensitive WB analysis of low abundant azide-tagged protein post translational modifications (PTMs) (Scheme 4


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1). Environmental-responsive polymers reversibly dissolve and self-assembly in aqueous solution in response to small environmental changes, such as temperature, pH and reduction-oxidation conditions16,17. This soluble polymer was prepared using thermo-responsive

isopropylacrylamide

(NIPAM)

and

acrylic

acid

for

copolymerization, which can be further functionalized with triarylphosphine and HRPs. The thermo-responsive polymer completely dissolves in aqueous solution at RT, which leads to facilitated interaction between the conjugated triarylphosphine groups and the azide-tagged target PTM-proteins on PVDF membrane. Next, the heavily loaded HRPs on the soluble thermo-responsive polymer provide amplified chemiluminiscent signal of the target PTM-protein band. More importantly, the signal is further improved by increasing the system temperature to above 40ºC and via temperature induced self-assemble of the thermo-responsive polymers. At elevated temperature, numerous polymer chains and conjugated HRPs aggregate within the confined area of the target PTM-proteins band and substantially

enhancing the

chemiluminiscent signal. As a result, two advantages can be achieved using this triarylphosphine and HRPs functionalized thermo-responsive soluble polymer. First, metabolic labeling with azide is available to a variety of PTMs, such as glycosylation, methylation, farnesylation and palmitic acid modification18-21, which broadens the application potential of this functionalized thermo-responsive polymer. One type of PTM donor substrate with azide modification is used for metabolic labeling in each test, so as to identify the specific type of PTM detected in the thermo-responsive polymer-based WB analysis. In this way, different kinds of PTM can be detected and 5



identified using the corresponding types of azide modified PTM donor substrate in different tests. The bioorthogonal reaction between azide and triarylphosphine is highly specific in complex sample22. Therefore, reduced non-specific protein binding and false-positive detection can be expected. Second, the soluble linear polymer carrier exhibits substantially reduced steric hindrance and facilitated interactions between the conjugated triarylphosphine and the azide-tagged PTM-proteins adsorbed on the PVDF membrane, compared with other solid/bulky carrier materials. Furthermore, the triarylphosphine modified polymers bound with the azide-tagged target PTM-proteins on the PVDF membrane may serve as aggregation centers during temperature induced polymer self-assembly, which leads to significantly increased amount of HRP surrounding the target PTM-proteins, while other unbound polymer assemblies can be removed by repeated washing. As a result, approximately 100 times more sensitive detection than commercial antibody was achieved using standard PTM proteins. We successfully applied this new functionalized thermo-responsive polymer for WB based analysis of O-GlcNAc, palmtic acid modified proteins and nascent proteins from HeLa cells, demonstrating the potential of this reagent in PTMs analysis.

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Scheme 1. Schematic overview of the thermo-responsive polymer based sensitive WB analysis of azide-tagged protein PTMs

Experiment Materials and reagents Bovine serum albumin (BSA), α-crystallin, N-isopropylacrylamide (NIPAM), acrylic acid, potassium persulfate, Thiamet G, lactalbumin, carbonic anhydrase, ovalbumin and β-galactosidase were obtained from Sigma (St. Louis, MO, USA). Luminol and peroxide detection solution, Ac3-6AzGlcNAc was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). NHS-triarylphosphine (NHS-Phosphine), Click-IT™ O-GlcNAc Enzymatic Labeling System, Click-iT® AHA (L-azidohomoalaine) and Click-IT™ Palmitic Acid Azide (15-Azidopentadecanoic Acid) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cytoplasmic and Nuclear fractionation kit and PNGase F was received from Cell Signaling Technology (Boston, MA, USA) and New England Biolabs (Beverly, MA, USA), respectively. The deionized water used in all of the experiments (resistance > 18 MΩ cm-1) was 7



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prepared using a Millipore purification system (Billerica, MA, USA).

Synthesis

of

thermo-responsive

poly-(N-isopropylacrylamide-co-acrylic

(poly-(NIPAM-AA)) and functionalization

acid)

with triarylphosphine and HRPs

Acrylic acid (0.12 mL), 0.8 g of N-isopropylacrylamide (molar ratio of AA : NIPAM= 1 : 4) and potassium persulfate (80 mg) were dissolved in 20 mL of degassed deionized water. pH of the solution was adjusted to 7.0 by adding 1 M NaOH. The mixture was allowed to react in a nitrogen environment at 70 ºC for 10 h with vigorous stirring. The obtained poly-(NIPAM-co-AA) copolymer was precipitated and recovered by addition of pure ethanol, freeze dried and stored in -20 ºC before usage.

Triarylphosphine and HRPs functionalization of poly-(PNIPAM-AA) was achieved by EDC-NHS coupling of the carboxyl groups of poly-(PNIPAM-AA) and the primary amine

groups

of

triarylphosphine-HRP

the

triarylphosphine-HRP

conjugates

were

prepared

conjugates. by

mixing

First, 1

mg

the of

NHS-triarylphosphine (NHS-Phosphine) dissolved in 0.2 mL of DMF with 20 mg of HRP in 0.8 mL of PBS buffer (pH 7.2). The mixture was kept agitating at RT for 2 h. The obtained triarylphosphine-HRP conjugates were purified by ultrafiltration (30 KDa cutoff). Next, 5 mg of poly-(NIPAM-co-AA) was dissolved in 1 mL of

MES

buffer (pH 5.0) containing 2 mM of EDC and 5 mM of NHS for 15 min. After adjusting the solution pH to 7.2 by adding 1 M NaOH, 20 mg triarylphosphine-HRP conjugates dissolved in 1 mL of PBS buffer (pH 7.2) with 2 mM of EDC and 5 mM of 8


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NHS was added and kept under RT for 2 h with vigorous mixing. The excess triarylphosphine-HRP was removed by ultrafiltration (100 KDa cutoff) of the solution. The amount of residual triarylphosphine-HRP in the flow-through solution from the ultrafiltration was quantified by measuring the UV absorption value at 280 nm and comparing this value with a calibration curve prepared by the UV absorption value at 280 nm of a series of triarylphosphine-HRP solutions of different concentrations. The loading amount of triarylphosphine-HRP on the thermo-responsive polymer was determined by subtracting the amount of residual triarylphosphine-HRP in the flow-through solution from the ultrafiltration from the initial amount of triarylphosphine-HRP used for conjugation with poly-(NIPAM-co-AA). Finally, the obtained poly (NIPAM-co-AA)-triarylphosphine-HRP (PNATH) was lyophilized and stored at 4 ºC until further use.

Characterization of PNATH and triarylphosphine-HRP The elemental composition of PNATH was analyzed by X-ray photoelectron spectroscopy (XPS) using a Kratos AMICUS system (Shimadzu, Japan). The hydrodynamic size was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, U.K.). Gel permeation chromatography (GPC) analysis was performed on a DAWN HELEOS system (Wyatt Technology, Santa Barbara, CA, USA). C4 reverse phase liquid chromatography analysis of HRP before and after NHS-Phosphine modification was performed on a L-3000 system (Rigol, Beijing, China). The morphology of PNATH was determined by an H-7650 9



(HITACHI, Japan) low to high resolution transmission electron microscopy (TEM).

Azide tagging of standard proteins and metabolic based azide tagging of complex protein sample 150 µg of α-crystallin, a standard O-GlcNAc protein was dissolved in the azide tagging buffer containing 0.1 mM of UDP-GalNAz, 50 mM of NaCl, 20 mM of HEPES, 5 mM of MnCl2 and 2% NP-40 at pH 7.9. After adding 15 µL of Gal-T1 (Y289L) enzyme, the solution was thoroughly mixed and incubated at 4 ºC overnight with gentle agitation. For metabolic based azide tagging of complex protein sample, HeLa cells were cultured in DMEM containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin at 37 ºC in a 5% CO2 incubator. The cells were cultured for 24 h after adding 200 µM Ac3-6AzGlcNAc, L-azidohomoalanine (AHA) or 15-Azidopentadecanoic Acid (APD) (final concentration) for metabolic labeling. Next, the cells were harvested and aspirated from the media with ice-cold phosphate buffer saline (PBS) for three times. For O-GlcNAc protein analysis, distinct cell fractions including cytoplasmic and nuclear/cytoskeletal were extracted from the obtained HeLa cells using corresponding cell fractionation kit. The cytoplasmic and nuclear/cytoskeletal fractions were combined and used for the subsequent O-GlcNAc proteins analysis (referred to as nucleocytoplasmic proteins). The nucleocytoplasmic proteins (2 µg/µL) was first treated with 100 units of PNGase F in 25 mM of ammonium bicarbonate at 37 ºC overnight for N-glycosylation removal to prevent interference from residual membrane/secreted glycoproteins. Next, the protein sample 10


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was precipitated using standard chloroform/methanol precipitation.

WB analysis of standard proteins and complex protein samples from HeLa cell using poly (NIPAM-co-AA)-triarylphosphine-HRP (PNATH) Perform SDS-PAGE and membrane transfer of the azide labeled proteins using standard procedures. Block the PVDF membrane with 1% BSA in PBS with 0.05% TWEEN 20 at RT for 1 hour and remove the excess reagents by repeated washing using PBS with 0.05% TWEEN 20. Incubate the PVDF membrane with 0.5 mg/mL PNATH in PBS with 0.05% TWEEN 20 for 4 hours at RT to allow the triarylphosphine group of PNATH to react with the azide-tagged proteins. After slowly increasing the system temperature to above 40 ºC and allowing the PNATH polymer to aggregate and self-assemble at the azide-tagged protein bands, the PVDF membrane was washed six times at 40 ºC using PBS with 0.05% TWEEN 20 to remove the non-specifically adsorbed PNATH polymer and self-assembly. Finally, the PVDF membrane was stained with luminol and peroxide detection solution at a 1:1 ratio in the dark at 40 ºC for 1 min and imaged using a GE ImageQuant LAS 500 system (GE Healthcare, Chicago, IL, USA).

Results and discussion Synthesis, Functionalization and Characterization of the thermo-responsive poly (NIPAM-co-AA)-triarylphosphine-HRP (PNATH) Synthesis of the thermo-responsive poly-(N-isopropylacrylamide-co-acrylic acid) 11



(poly-(NIPAM-AA)) and functionalization with triarylphosphine-HRPs via free radical polymerization and EDC-NHS coupling was described in the experiment section and in Figure 1. Gel permeation chromatography (GPC) analysis was performed and the molecular weight (Mn) of PNATH was determined to be 138 KDa (Mw/Mn=1.333, Figure S1, Supporting Information). The poly-NIPAM region of PNATH exhibits sensitive temperature induced morphology changes from linear chains to collapsed globules and eventually large scale intermolecular self-assembly of numerous polymer chains16. The poly-acrylic acid region carrying densely packed carboxyl groups can be further functionalized with triarylphosphine-HRPs. As a result, a two-level signal amplification in WB analysis can be achieved. The signal is first amplified by the multiple triarylphosphine-HRPs carried by each PNATH polymer chain that is covalently coupled to the azide-tagged target protein. Second, after increasing the system temperature above the lower critical solution temperature (LCST) of PNATH, the amount of triarylphosphine-HRP and the corresponding signal is further amplified by the intermolecular self-assembly of numerous PNATH polymer chains around each polymer chain that is covalently coupled to the azide-tagged proteins on the PVDF membrane.

Figure 1. Synthesis route of poly (NIPAM-co-AA)-triarylphosphine-HRP (PNATH). 12


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Triarylphosphine-HRPs are synthesized by reaction between the NHS ester of NHS-Phosphine and the primary amine of HRP. Considering that there are six lysine residues and one N-terminal with primary amine in the sequence of HRP and given the high reactivity of NHS ester, a high ratio of triarylphosphine modification on HRP can be expected, which facilitates the conjugation of Triarylphosphine-HRPs with the low abundant azide-tagged target proteins on the PVDF membrane. The obtained triarylphosphine-HRP was characterized by C4 reverse phase liquid chromatography. As shown in Figure S2 (Supporting Information), HRP shows longer retention time after triarylphosphine modification due to the high hydrophobicity of the triarylphosphine groups. The new peaks at 10.4 min shows a peak width similar tto that of the original HRP peak at 10.2 min with no obvious peak spreading indicating uniform triarylphosphine modification of the HRP molecules. Almost no unmodified residual HRP can be identified at 10.2 min demonstrating high efficiency of triarylphosphine modification of HRP. Next, the obtained triarylphosphine-HRPs were conjugated with the acrylic acid groups of the PNIPAM-AA copolymer and the obtained poly (NIPAM-co-AA)-triarylphosphine-HRP (PNATH) was subjected to further characterization. X-ray photoelectron spectroscopy (XPS) was used to verify successful attachment of the triarylphosphine groups. After modification of PNIPAM-AA with triarylphosphine-HRP, a characteristic phosphorus absorption peak at ∼ 130.5 eV is observed in the XPS spectrum (Figure S3, Supporting Information) of the copolymer. In contrast, no corresponding peak is found for the PNIPAM-AA 13



copolymers without triarylphosphine-HRP modification. The loading amount of triarylphosphine-HRP on PNATH was determined to be 1.74±0.06 mg/mg. Given that thethe molecular weights of PNATH and HRP are 138 and 44 KDa, respectively, approximately 5.45 triarylphosphine-HRP molecules are attached to each PNATH chain on average. The high triarylphosphine-HRP content of PNATH copolymer may effectively facilitate the reaction between PNATH and the low abundant azide-tagged target proteins in the complex sample via bioorthogonal Staudinger ligation and leading to amplified HRP based chemiluminiscent signal.

Thermo-responsive behavior and hydrodynamic size of PNATH at different temperatures was evaluated using dynamic light scattering (DLS). Poly-NIPAM based thermo-responsive polymer chains exhibit inverse solubility upon heating. In Figure 2, the hydrodynamic size of PNATH measured by DLS shows a sharp increase from less than 15 nm to more than 130 nm within a 10 ºC range, indicating a dramatic change in the microcosmic morphology of PNATH from dissolved linear polymer chains to aggregated self-assembly of nanometer-sized particles. The dissolved or aggregated PNATH collected at RT or 40 °C was further characterized by transmission electron microscopy (TEM) shown in Figure S4, Supporting Information. PNATH agglomerates into round particles at temperature above its LCST with diameters of about 100 nm, which is consistent with those obtained using DLS. At temperature blow its LCST, PNATH adopts a hydrated linear conformation and is completely dissolved to form a transparent homogenous solution (inserted picture of Figure 2) 14


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providing a soluble matrix to facilitate the reaction between the triarylphosphine of PNATH and the azide of the target protein on the PVDF membrane. Upon heating the solution to above 40 °C, PNATH polymer chains rapidly change to a globular dehydrated

microstructure

and

further

self-assemble

into

nanometer-sized

agglomerations containing numerous PNATH polymer chains and a large amount of HRP, therefore a substantially enhanced signal in the WB analysis can be expected.

Figure 2. DLS analysis of the hydrodynamic diameters of PNATH under different temperature. Inserted: picture of PNATH solution under different temperature.

Feasibility, sensitivity and specificity of PNATH based WB analysis of O-GlcNAc modified proteins The feasibility of using PNATH for WB analysis of azide-tagged PTM-protein was demonstrated using azide-tagged O-GlcNAc proteins as a model sample. O-GlcNAcylation is a highly dynamic PTM that substoichiometricly cycles on and off serine

and

threonine

residues

of

low-abundance 15


regulatory

proteins23,24.


O-GlcNAcylation plays critical roles in signal transduction25, transcriptional control26, and protein degradation27. Abnormally regulated O-GlcNAcylation has been found to have close correlation with many major human diseases, such as diabetes24,28, cancer29,30 and Alzheimer disease31. Identification and quantification of O-GlcNAc proteins though highly challenging, is a prerequisite for deciphering their roles in disease occurrence and development. Unlike phosphorylation or acetylation, for which a wide range of pan and site-specific antibodies are available, studies of O-GlcNAc modification are hampered by the lack of effective tools for the detection and quantification of this PTM. Therefore, a new method that provides sensitive and specific detection of O-GlcNAcylation may greatly facilitate the biological function study of this PTM. PNATH-based WB analysis of O-GlcNAc modified proteins was first evaluated using mixture of α-crystallin (a standard O-GlcNAc protein) and three non-O-GlcNAc proteins of various molecular weights (BSA, ovalbumin, and carbonic anhydrase). α-crystallin was treated with azide modified UDP-GalNAz and Gal-T1 (Y289L) enzyme for in vitro azide tagging of O-GlcNAc. As shown the gel image in Figure 3, lane 2 is the protein mixture after Coomassie brilliant blue staining. The four proteins are clearly visible in the gel from 20 to 66 KDa. However, only α-crystallin can be detected in lane 3 using PNATH-based WB analysis with no trace false-positive signal from the three non-O-GlcNAc proteins. Though α-crystallin can also be detected by commercial anti-O-GlcNAc antibody (lane 4), intensity of the protein band is much weaker than that of PNATH. The detection sensitivity of PNATH was determined using a series of α-crystallin with different loading amount 16


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from 2 µg to 0.01 µg. As shown in Figure S5 (Supporting Information), intensity of the protein bands reduces with decreasing amount of α-crystallin and the protein band is still clearly visible when using 0.01 µg of α-crystallin. Given that there is only one major modification site and given the low stoichiometry of O-GlcNAcylation (~10%) of α-crystallin32, the actual detection sensitivity of using PNATH is approximately 1 ng. Further comparison of lane 5 in Figure S5a and lane 7 in Figure S5b reveals similar gray levels and intensities of the two protein bands, while PNATH is 100 times more sensitive than the antibody.

Figure 3. PNATH based WB analysis using mixture of the standard O-GlcNAc protein and non-O-GlcNAc proteins.

To further challenge PNATH, highly complex protein samples from cell lysate were used. O-GlcNAc modified proteins in Hela cells were metabolically tagged with azide using Ac36AzGlcNAc as the substrate sugar donor in cell culture18. To remove possible contaminant N-glycoproteins and mucin typed O-linked glycoproteins, PNGase F treated nucleocytoplasmic protein was used and separated by SDS-PAGE. 17



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PNATH based WB analysis was performed as described in the experimental section. In Figure 4a, different amount of nucleocytoplasmic protein from 10 to 0.1 µg was loaded in lane 2 to 6. Intense protein bands are visible in lane 3 with 10 µg of nucleocytoplasmic

protein,

but

no

bands

can

be

observed

in

lane

2

(nucleocytoplasmic proteins without azide tagging) with the same amount of nucleocytoplasmic protein. Lowering the protein loading amount results in a similar number of protein bands on the PVDF membrane with faint gray level. The above results demonstrate that highly specific bioorthogonal reaction between the triarylphosphine modified PNATH and the azide-tagged O-GlcNAc proteins was achieved with almost no non-specific adsorption of PNATH on non-azide labeled proteins. Considering that the level of O-GlcNAc protein is usually less than 1% of the total nucleocytoplasmic protein33, the high sensitivity of PNATH based WB analysis is still valid in complex protein samples and is capable of detecting low ng level of O-GlcNAc protein with a dynamic range of two orders of magnitude. The PNATH based WB analysis of O-GlcNAc protein was further evaluated by comparing this method with PNATH based detection without heating treatment. Similar detection patterns were observed but with noticeably reduced sensitivity, as shown in Figure 4b. The bands are barely detectable when the protein loading amount is reduced to 5 µg. We attribute the difference in detection sensitivity to the unique temperature induced self-assembling behavior of PNATH. Since each azide tagged O-GlcNAc is capable of conjugation with only one PNATH polymer chain carrying a few HRPs, the signal amplification is limited. However, the temperature induced self-assembly of PNATH 18


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leads to agglomeration of numerous polymer chains with conjugated HRPs around the PNATH chain that is coupled to the PVDF membrane adsorbed azide-tagged O-GlcNAc protein. Furthermore, other polymer assemblies without the PNATH chain coupled to the PVDF membrane can be removed by repeated washing to avoid false-positive signals. In this way, greatly increased amount of PNATH and HRPs within the O-GlcNAc protein bands results in substantially enhanced detection sensitivity, while maintaining good specificity.

Figure 4. PNATH based WB analysis of O-GlcNAc proteins from HeLa cells with (a) or without heating treatment (b). + and – refer to nucleocytoplasmic proteins with or without azide labeling.

To further assess the detection selectivity and reliability of the PNATH based WB detection of O-GlcNAc proteins, O-GlcNAcase (OGA) inhibitor (Thiamet G) treated HeLa cells were used and compared with HeLa cells without inhibitor treatment. OGA is the enzyme responsible for the removal of O-GlcNAc modification from 19



Page 20 of 25

proteins in vivo, therefore elevated O-GlcNAc level is expected in the OGA inhibitor treated cells. As shown in Figure 5a, the number of protein bands and the corresponding gray levels increase in lane 3 using nucleocytoplasmic proteins extracted from OGA inhibitor treated cells compared with that observed using cells without

inhibitor

treatment

in

lane

2.

The

consistent

enhancement

of

O-GlcNAcylation level and signal intensity in the PNATH based WB detection demonstrates the reliability and selectivity of this new detection method. Further analysis of the OGA inhibitor treated and non-treated samples using a commercial anti-O-GlcNAc antibody (RL2) shows similar protein band patterns, but with much lower signal intensity with the same amount of protein loading (Figure 5b), indicating obviously improved sensitivity using PNATH. Interestingly, PNATH exhibits a distinct advantage in the detection of low molecular weight O-GlcNAc proteins. More protein bands were detected in the 10-50 KDa region. The possible bias of RL2 towards high molecular weight protein targets is not unexpected. Presumably, this phenomenon correlates with the high molecular weight nuclear pore protein antigen used for the development of RL2, the anti-O-GlcNAc antibody. This bias toward antigen proteins/motif sequences is a common problem for PTM specific antibodies developed using protein/peptide-based antigens, however not an issue for PNATH, since the recognition by PNATH is solely dependent on azide-triarylphosphine based Staudinger ligation.

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Figure 5. WB analysis of O-GlcNAc proteins from HeLa cells with (+) or without (–) OGA inhibitor (thiamet G) treatment using PNATH (a) or anti-O-GlcNAc antibody (RL2) (b).

To expand the application potential of the PNATH based WB analysis, two more tests were carried out. L-azidohomoalanine (AHA) or 15-Azidopentadecanoic Acid (APD) was fed to HeLa cells and metabolically incorporated into proteins in vivo. AHA is an azide modified amino acid analog that can be used to characterize newly synthesized proteins. APD is commonly used to study palmitoylation attached to cysteine residues and less frequently to serine and threonine residues of proteins. As shown in Figure S6a (Supporting Information), AHA labeled newly synthesized proteins can be clearly detected by PNATH-based WB analysis. As expected, introduction of additional methionine during cell culture effectively inhibits the incorporation of AHA by 21



Page 22 of 25

proteins and noticeably reduces the number of protein bands & intensity detected. Similar results were obtained for protein palmitoylation study using APD for metabolic labeling. Palmitoylated proteins obtained from APD treated HeLa cells can be also detected by PNATH in Figure S6b (Supporting Information). Removal of palmitoylation by NH4OH treatment leads to almost complete abolishment of the corresponding

protein

bands,

demonstrating

efficient

detection

of

protein

palmitoylation via specific Staudinger ligation and signal amplification by the thermo-responsive PNATH.

Conclusion In

this

work,

we

synthesized

triarylphosphine

and

HRPs

functionalized

thermo-responsive and self-assembling polymers for sensitive western-blot (WB) analysis of low abundant azide-tagged PTMs. This thermo-responsive polymer complete dissolves in aqueous solution at RT, which leads to facilitated interaction between the polymer conjugated triarylphosphine and the azide-tagged target PTM-proteins on PVDF membrane. More importantly, the polymer chains that are coupled to the PVDF membrane serve as aggregation centers during the temperature induced polymer chains self-assembly. The sub-micrometer sized polymer agglomerates lead to a significantly increased amount of HRP around the target PTM-proteins and enhanced the chemiluminiscent signal. Successful application of the new functionalized thermo-responsive polymer for WB-based analysis of azide-tagged O-GlcNAc, palmtic acid modified proteins and nascent proteins from 22


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HeLa cells demonstrates the potential of this strategy in protein PTM studies, even though with limitations including not capable of directly detecting native PTMs from cell, tissue or blood samples.

Supporting Information The Supporting Information including additional experimental details, data, and data analysis is available free of charge on the ACS Publications website.

Acknowledgements This study was supported by National Key Program for Basic Research of China (No. 2016YFA0501403,

2017YFC0906700,

2017YFA0505002,

2014CBA02001),

National Natural Science Foundation of China (No. 21235001, 21405175 and 21675172), BPRC-Tianjin Baodi Hospital Joint Center Grant (TMRC2014Z03), Innovation Project (16CXZ027) and Beijing Science and Technology Plan Project (No. Z161100002616036).

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Sensitive Western-Blot Analysis of Azide-Tagged Protein Post

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