Selective Isolation of Myosin Subfragment-1 with a DNA

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Selective Isolation of Myosin Subfragment-1 with a DNA-Polyoxovanadate Bioconjugate Qing Chen, Xue Hu, Dan-Dan Zhang, Xuwei Chen, and Jian-Hua Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00597 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Bioconjugate Chemistry

Selective Isolation of Myosin Subfragment-1 with a DNA-Polyoxovanadate Bioconjugate Qing Chen, Xue Hu, Dan-Dan Zhang, Xu-Wei Chen*, Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China *Corresponding author. E-mail: [email protected] (X.-W. Chen); [email protected] (J.-H.Wang).

Abstract: The bioconjugation of a polyoxometalate (POMs), i.e., dodecavanadate (V12O32), to DNA strands produces a functional labeled DNA primer, V12O32-DNA. The grafting of DNA primer onto streptavidin-coated magnetic nanoparticles (SVM) obtains a novel composite, V12O32-DNA@SVM. The high binding-affinity of V12O32 with the ATP binding site in myosin subfragment-1 (S1) facilitates favorable adsorption of myosin, with an efficiency of 99.4% when processing 0.1 mL myosin solution (100 µg mL-1) using 0.1 mg composite. Myosin adsorption fits Langmuir model, corresponding to a theoretical adsorption capacity of 613.5 mg g-1. The retained myosin is readily recovered by 1% SDS (m/m), giving rise to a recovery of 58.7%. No conformational change is observed for myosin after eliminating SDS by ultrafiltration. For practical use, high-purity myosin S1 is obtained by separation of myosin from the rough protein extract from porcine left ventricle, followed by digestion with α-chymotryptic and further isolation of S1 subfragment. The purified myosin S1 is identified with matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry, giving rise to a sequence coverage of 38%.

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INTRODUCTION Heart failure, a leading cause of death, affects millions of people around the world and the studies of its nosogenesis are focused on the muscle proteins. Cardiac myosin is the major force-generating motor protein of cardiac muscle. The contractile function of muscle can be modulated by the ATPase activity of myosin, which could be regulated and used to treat systolic heart failure1,2. Myosin comprises two unique structures, i.e., a globular double-headed structure subfragment-1 (S1) containing ATPase and F-actin binding sites, and a long flexible coiled tail rod3. The myosin S1 is involved in the mutational analyses in a myosin structure study, e.g., the autosomal dominant mutations in human myosin heavy chain genes cause a variety of cardiac myopathies. For the purpose of discovering new mutations, the investigations on myosin S1 of various origins have been conducted by using both biochemical kinetics and in vitro motility approaches in the latest studies4,5. Considering that myosin heavy chain genes can express various isomers and mutants, it is highly important to separate myosin S1 from biological samples to identify the difference which may cause lesions or affect the behavior of the cells. Polyoxometalates (POMs) are a large family of metal-oxygen clusters of the early transition metals in high oxidation states, most commonly vanadium (V), molybdenum (VI) and tungsten (VI)6,7. Due to the gigantic nanoscale molecular structures and an unmatched range of physical and chemical properties, POMs are referred as metal oxide building blocks and received considerable interests8,9. POMs present controllable shape and size, oxo-enriched surfaces and highly electronegative


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properties, make them suitable to act as catalysts, adsorbents, photo-electronic materials, magnetic materials and anti-tumor drugs10-13. Polyoxovanadates (POVs), as an important subfamily of POMs, exhibit unprecedented structural diversity, largely attributed to their versatile redox activities and the variable coordination geometries of the vanadium centers which can adopt VO4 tetrahedra, VO5 square pyramids, VO6 octahedra as well as their mixtures. Therefore, POVs can serve as excellent building units in the formation of nanoscale or supramolecular architectures14-16. Decavanadates (V10O28), as the predominant polyoxovanadates, have recently attracted extensive attentions due to their medicinal and biochemical behaviors17-19. The protonation of decavanadates allows them to interact with proteins and biological molecules. Mammalian cell-surface NTPDase enzymes are inhibited by the decavanadate cluster, wherein the in situ formed V10O28 occupies the active-site cleft of NTPDase20. The V10O28 unit produces non-competitive inhibition of the F-actin stimulated subfragment-1 (S1) ATPase activity due to the high-affinity binding with the back door of the catalytic site and the conserved region of the phosphate binding-loop (P-loop) of S121,22. POMs have also been used in protein crystallography due to their selectivity in binding with specific regions of proteins23-25. Functionalization via covalently grafting of organic functional groups allows POMs to tune their redox ability, acidity and solubility, together with a potential modulation of essential features, e.g., stability, bioavailability and recognition capability26,27. A lot of POMs containing compounds have been prepared with POMs retaining their structural integrity, including organonitrogen28, organosilyl29,



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organophosphonyl30 and oxocarbon31 derivatives of POMs. The heteropolytungstates [α2-P2W17O61(SnR)]7 has been reported with an alkyltin group with promising antitumor activities32. Sn (IV) directs the cyclization to specific oxo-ligands due to the stereoelectronic constraints and the probably increased nucleophilicity33. To the best of our knowledge, much less effort is devoted to the bioconjugation of POMs despite their known bioactivity34. It is indicated that POM-drug conjugates could be an efficient approach to covalently graft a bioactive ligand, e.g., amantadine, onto POMs and enhance their application in clinical cancer treatment35. The self-assembly of hierarchical structures from dopamine and POMs shows an excellent pH-responsive release of doxorubicin and is used for oral drug delivery36. Peptides grafted onto POMs shows a potential for POMs to serve as unnatural building units in solid phase peptide synthesis37. Recently, biofunctionalization of POMs with DNA primers facilitates electrochemical detection of PCR products, demonstrating its potential applications in bioanalytical field38. Herein, we present for the first time the incorporation of alkyltin group into a classical cage-like POV framework. The generated V12O32[Sn(CH2)2CO2H]4- is used to link to a 5’-NH2 terminated 21-mer DNA forward primer through amide coupling, and V12O32-labeled DNA grafting onto streptavidin-coated magnetic nanoparticles (SVM) produces a novel composite, V12O32-DNA@SVM. It exhibits favorable adsorption selectivity towards myosin. The isolation of S1 from myosin is achieved via V12O32-DNA@SVM adsorption after its digestion by α-chymotrypsin. Further, practical applications for the isolation of myosin S1 from porcine heart is


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demonstrated by a cascade extraction procedure, that is, myosin is first isolated from rough protein extract from porcine left ventricle, and myosin S1 is then collected after digestion of myosin with α-chymotryptic.

RESULTS AND DISCUSSION Characterization of the V12O32-DNA conjugation. For the functionalization of V12O32, carboxylic acid is grafted by the reaction of bowl-shaped dodecavanadate (V12O32) with Cl3Sn(CH2)2CO2H, where the oxygen rich ‘lip’ of the V12O32 bowl provides a good binding site for organotin RSnCl3 and the high affinity of RSnCl3 towards oxygen donors renders them a good match16,39. The aminated oligonucleotide 5’-NH2-(CH2)6PO3-ATTACAATGGCAGGCTCCAGA-biotin-3’ is dissolved in a small amount of ddH2O in the presence of a triethylamine and excessive V12O32, for the compensation of undesired reaction of carboxylic acid on V12O32-COOH. DMSO as the reaction solvent facilitates complete dissolution of the reactants and the amide coupling reaction between the aminated DNA and V12O32-COOH (Scheme 1). The V12O32-DNA conjugate is then characterized by vibrational spectroscopy as illustrated in Figure 1.

Scheme 1. The fabrication scheme for V12O32-DNA@SVM composite.



FT-IR spectra in Figure 1A indicates that the adsorptions within 1000-650 cm-1 are assigned to V=O and V-O stretching vibrations for V12O3240. For V12O32-COOH, its FT-IR spectrum shows the characteristic absorptions of V12O32 while no splitting of the V-O and V=O stretching vibration is observed, indicating that the lacuna of V12O32 is filled41. The characteristic adsorptions of DNA are clearly identified at 1697, 1636, 1540, 1372, 1219, 1058 and 1013 cm-1. It is seen that FT-IR spectrum of the V12O32-DNA bioconjugate exhibits the characteristic absorptions of both V12O32 and DNA. In addition, the absorption bands at 1698, 1651 and 1603 cm-1 demonstrate the formation of an amide bond between the carboxylic group on the V12O32-COOH and amino group of NH2-terminated DNA38. Raman spectrum of V12O32-DNA in Figure 1B contains the typical absorption bands of V=O at 1008 cm−1 in addition to those expected for DNA at 575, 779, 1094, 1337, 1574, 1572 cm-1. The above observations well demonstrated the conjugation of V12O32 with DNA.

V12O32

POM cluster

A

B

POM cluster 1008 1008

V12O32-COOH

1483

993 861

(1642, COOH)

V12O32-DNA

(1697, C=O) from C, G, T

2000

760

(1483, -CH2-)

(1698, 1651, 1603 POM-CONH-DNA)

DNA

Intensity Intensity (a.u.) (a.u.)

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Intensity(a.u.) Intensity (a.u.)

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(1058, asPO2)

(1372, -CH2-) (1219, asPO2) (1540, C=C) (1636, C=N)

1550

1100

Wavenumber(cm-1 )

(1013, DNA backbone)

V12O32 (1465, -CH2-)

V12O32-COOH (1572,A) (1574,A,T,C) (1337,A)

V12O32-DNA(575, G) (779, T,C)

(1094, bk(PO2-st)

DNA

650

100

400

700

1000

1300

1600

Wavenumber (cm-1-1) ) Wavenumber(cm

Figure 1. FT-IR (A) and Raman (B) spectra of V12O32, DNA, V12O32-COOH and V12O32-DNA conjugate. FT-IR spectra: sample amount, 0.1 mg; resolution, 2.0 cm-1. Raman spectra: sample amount, 0.1 mg; scan range, 100-1600 cm-1.


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Characterization of the V12O32-DNA@SVM composite. The oligonucleotide 5’-NH2-(CH2)6PO3-ATTACAATGGCAGGCTCCAGA- biotin-3’ is biotinylated. Thus, V12O32-DNA conjugate generates strong biotin-streptavidin interaction with streptavidin-coated magnetic nanoparticle (SVM) by mixing them (Scheme 1). Magnetite (Fe3O4) nanoparticles are obtained by a solvothermal approach with ethylene imine polymer as the protective agent for preventing the particles from aggregation42. SEM images in Figure 2A reveal that the uncoated magnetite particles are spheres with an average size of ca. 300 nm. After coating with glutaraldehyde and streptavidin, the size of the magnetic particles is increased to ca. 400-500 nm (Figure 2B). Figure 2C clearly indicated that the grafting of V12O32-DNA further increases the size of V12O32-DNA@SVM composite to ca. 600 nm.

Figure 2. SEM images of the magnetic nanoparticles Fe3O4-NH2 (A), the streptavidin-coated magnetic nanoparticles (B) and the V12O32-DNA@SVM composite (C). Sample amount, 0.1 mg; resolution: ≤0.8 nm; acceleration voltage: 15 kV.

Protein adsorption behaviors by V12O32-DNA@ SVM. Myosin is best known



for its role in muscle contraction and cardiac myosin is the major force-generating motor protein of cardiac muscle. The separation of myosin from cardiac muscle is necessary for elucidating the mechanisms of heart diseases. To evaluate the affinity of V12O32-DNA@SVM composite towards various proteins, five proteins, i.e., myosin (MYO), bovine serum albumin (BSA), cytochrome c (cyt-c), transferrin (TRF) and myoglobin (MYB) are chosen as models. 0.1 mg of V12O32-DNA@SVM composite is added into 0.1 mL of the protein solutions (100 µg mL-1) and the mixture is shaken vigorously for 30 min at room temperature to facilitate the adsorption of proteins onto the surface of composites. Their adsorption efficiencies within pH 4-10 are shown in Figure 3A. It is obvious that V12O32-DNA@SVM composite exhibits excellent binding performance towards MYO at a wide pH range with an adsorption efficiency of >90%. In the meantime, however, the adsorption of other coexisting proteins is rather limited within the whole pH range tested. A

80

MYO TRF BSA cyt-c MYB

60 40 20 0 3

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pH

8

9

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100

Adsorptionefficiency efficiency(%) (%) Adsorption

100

Adsorptionefficiency efficiency(%) (%) Adsorption

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80

MYO TRF BSA cyt-c MYB

60 40 20 0 -100

100

300

500

700

900

1100

NaCl concentration (mM)

Figure 3. The effect of pH (A) and ionic strength (B) on the adsorption behaviors of MYO, TRF, BSA, cyt-c and MYB with V12O32-DNA@SVM composite as adsorbent. Protein solution: 100 µg mL-1, 0.1 mL; V12O32-DNA@SVM composite: 0.1 mg; adsorption time: 30 min.


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Generally, the coexisting salts exhibit profound effects on the processes of protein adsorption43. In the present case, the effect of ionic strength on the adsorption of the five proteins is investigated by the addition of certain amount of NaCl into the protein solution. Figure 3B shows a decrease on the adsorption efficiency of the other four proteins with the increase of ionic strength up to a NaCl concentration of 250 mmol L-1, and thereafter a slight increase of the adsorption efficiency is observed when further increasing the ionic strength. In general, with the increase of ionic strength or salt concentration, the cationic and anionic species tend to be attracted on the protein molecule surface to a saturated condition and thus the charge on protein molecules approaches to a constant level. At this point, the screening effect, ionic affinity with the protein surface, dominates and reduces the repulsive double layer force, leading to a salting-out effect43. It should be noticed that the change of ionic strength within a wide range causes very limited variation on the adsorption of MYO, i.e., an adsorption efficiency of >80% still remains at an ionic strength of 1000 mmol L-1. It is well-known that the salting-out effect of myosin occurs at a much higher salt concentration than the conventional proteins44. This behavior is obviously most attractive and promising for the selective isolation of myosin from real biological samples wherein high concentration of salts is generally encountered. That is to say, it is not necessary to control the ionic strength and/or pH value of the sample solution for practical adsorption of MYO from biological samples, while ddH2O is adopted for the preparation of protein solutions. Myosin molecule is composed of head, neck, and tail domains. The globular



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head is well conserved containing the binding sites for ATP and actin, which is responsible for ATPase activity during the process of muscle contraction. The ATP binding site contains the structure of β-α-β composing of β-sheet, α-helix and β-sheet. The β-sheet in the structure of all myosin heavy chains contains the sequence of Gly-Glu-Ser-Gly-Ala-Gly-Lys-Thr45. Lys (lysine) in the sequence can interact with alpha-phosphate of the bound ATP. The structure of vanadate is very similar to that of phosphate, and vanadate can occupy the ATP binding site in myosin subfragment-1 (S1) which inhibits the actin-stimulated myosin ATPase activity noncompetitively46. The vanadate is contacted by Lys from the domains which are rather alike a certain shape complementarity that favors binding in a specific orientation20. That is, V10O32 interact with the ATP binding site and V12O32-DNA@SVM composite as an adsorbent is similar to the fishhook that can selectively adsorb myosin. Figure 4A illustrates the dynamic adsorption isotherm of MYO onto the V12O32-DNA@SVM composite at room temperature within a range of 50-600 mg L-1. It fits well with Langmuir adsorption model as expressed in the following equation, with Ce (mg L-1) as the protein concentration, Q* (mg g-1) as the amount of adsorbed protein at equilibrium, Qm (mg g-1) as the adsorption capacity and Kd as the adsorption constant. By fitting the experimental data to the Langmuir adsorption model, an adsorption capacity of 613.5 mg g-1 is derived. These observations illustrated that V12O32-DNA@SVM composite can isolate myosin with high selectivity and a favorable adsorption capacity. Q* =

Qm × C e K d + Ce


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70

A

600

B 60

Elution efficiency (%)

Adsorbed myosin (mg g-1)

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450

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Myosin concentration (mg L-1)

H2O

BR

BR

Tris

Imi

SDS

pH 5

pH 9

50mM

500mM

1%

Figure 4. (A) The adsorption isotherm of MYO by the V12O32-DNA@SVM composite; (B) The recoveries of the adsorbed MYO from V12O32-DNA@SVM composite using various buffers as stripping reagents. Protein solution: 0.1 mL; V12O32-DNA@SVM composite: 0.1 mg; adsorption time: 30 min. In practice, it is important to transfer the adsorbed/isolated protein species from the adsorbent to an appropriate aqueous medium to facilitate further biological studies. In this respect, various buffer solutions, e.g., ddH2O, BR (pH 5.0), BR (pH 9.0), 50 mmol L-1 Tris, 500 mmol L-1 imidazole and SDS (1%, m/m), have been investigated for stripping/recovery of the adsorbed MYO from V12O32-DNA@SVM composite. As illustrated in Figure 4B, a favorable recovery of 58.7% for MYO is achieved by employing a SDS (1%, m/m) solution as the stripping reagent. It is also important to evaluate whether there is denaturation for the myosin recovered from the V12O32-DNA@SVM composite by use of SDS as the stripping reagent. This is done by investigating the conformational change of MYO by means of far-UV circular dichroism (CD) spectra (Figure 5). In an aqueous medium (deionized water), MYO exhibits two clear negative bands at 210 nm and 225 nm



attributed to the α-helix, as illustrated in Figure 5a. After adsorbed onto the V12O32-DNA@SVM composite, the recovered MYO by stripping with a SDS solution shifts the corresponding bands to 209 nm and 220 nm, respectively (Figure 5b). This clearly indicates a structural and/or conformational change for MYO during the adsorption and desorption processes. In order to elucidate whether this change is caused by the adsorption of V12O32-DNA@SVM composite or by the stripping of SDS, CD spectra of MYO stripped into SDS (1%, m/m) solution (Figure 5b) and the pure MYO directly dissolved in a same SDS solution are recorded for comparison (Figure 5c). It is obvious that their spectra are almost identical, indicating that the conformational change of MYO is most probably caused by the SDS solution. For the purpose of determining whether the conformational change of MYO is reversible, the eluate is subject to ultrafiltration using a 3 kDa centrifugal filter to remove SDS, and CD spectrum is thereafter recorded. Figure 5d illustrated that after removal of SDS, an identical CD spectrum is observed for MYO as that for pure MYO dissolved in deionized water without treatment by V12O32-DNA@SVM composite in Figure 5a, showing two characteristic bands at 210 nm and 228 nm. These results clearly indicate that V12O32-DNA@SVM composite exhibits favorable biocompatibility. 50 35

Ellipticity (mdeg)

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20 5 190 -10

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d 230

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Wavelength (nm)


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Figure 5. Soret region CD spectra of MYO. (a) 100 µg mL−1 MYO in deionized water; (b) MYO stripped into 1% SDS aqueous solution; (c) MYO directly dissolved in 1% SDS aqueous solution; (d) MYO in the eluate after removal of SDS by use of ultrafiltration with a 3 kDa centrifugal filter.

Separation and purification of myosin S1 by V12O32-DNA@SVM. Myosin digestion is preformed in a medium containing 0.05 mol L-1 KCl, 20 mmol L-1 Tris-HCl (pH 7.0) and 1 mmol L-1 EDTA, using TLCK-treated α-chymotrypsin to myosin ratio of 1/50 (w/w) at 10°C. Proteolysis is terminated by the addition of 0.5 mmol L -1 PMSF (final concentration)38. Typically, 0.1 mg V12O32-DNA@SVM composite is added into 100 µL digestion solution. After incubation for 30 min at room temperature, the mixture undergoes separation by a magnet and the supernatant is collected. Then, the separated V12O32-DNA@SVM composite is pre-washed with 500 mmol L-1 imidazole solution to remove the nonspecific adsorption proteins and immersed in 50 µL SDS (1%, m/m) solution for shaking 20 min to recover the adsorbed S1. SDS-PAGE assay is performed by using 12% polyacrylamide resolving gel and 5% polyacrylamide stacking gel with standard discontinuous buffer system. The purification of myosin S1 is confirmed in Figure 6. Lane 1 is the myosin solution with a main band at 200 kDa. The digestion pattern of myosin is shown in Lane 2 with the band of S1 at 96 kDa. In Lane 3, the band of S1 becomes weaker after adsorption by V12O32-DNA@SVM composite, and the other bands are still observable. After



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recovery by 1% SDS solution, only a single band is observed at 96 kDa, which is safely assigned to myosin S1 (Lane 4). This result clearly illustrates the interaction between V12O32-DNA@SVM composite and myosin.

Figure 6. SDS-PAGE assay results of the purification of myosin S1. Lane 1: 500 µg mL-1 myosin solution; Lane 2: the digestion solution of myosin using TLCK-treated α-chymotrypsin of 1/50; Lane 3: the digestion solution after treating with V12O32-DNA@SVM composite; Lane 4: The recovered solution by stripping with 1% SDS solution.

Separation and identification of myosin S1 from porcine heart by cascade extraction with V12O32-DNA@SVM. A two-step cascade extraction procedure is carried out to separate myosin and subfragment S1 (as illustrated in Scheme 2). Typically, myosin is separated from the rough protein extract of the porcine left ventricle with V12O32-DNA@SVM composite as adsorbent. Afterwards, myosin is recovered from the surface of the V12O32-DNA@SVM composite with a SDS (1%, m/m) solution. The collected myosin is further purified by elimination of SDS with


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ultrafiltration, followed by digestion with α-chymotryptic. Then, S1 is isolated and purified from the digestion solution with V12O32-DNA@SVM composite by following a same extraction procedure.

Scheme 2. The cascade extraction of myosin S1 from porcine heart with V12O32-DNA@SVM composite as adsorbent. Protein solution: 0.1 mL; V12O32-DNA@SVM composite: 0.1 mg; adsorption time: 30 min; 1% SDS solution ,0.1 mL; elution time, 20 min.

Figure 7A shows SDS-PAGE results of the purification of myosin and S1 from porcine heart. Lane 1 is the porcine heart protein extract from the porcine left ventricle which contains hundreds of proteins from 10 to 200 kDa, and the band at 200 kDa is assigned for myosin. The adsorption by V12O32-DNA@SVM composite causes a little change for the band of myosin due to the complex composition and high concentration of porcine heart protein extract (Lane 2). The stripping with SDS (1%, m/m) makes the band of myosin at 200 kDa clearly observable (Lane 3). After SDS elimination and α-chymotryptic digestion, a few bands are clearly shown within a



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range of molecular weight from 20.1-200 kDa (Lane 4). These protein bands are still observable in the residual solution after treatment by V12O32-DNA@SVM composite, while the intensity of the band for S1 (96 kDa) becomes much weaker (Lane 5). The further adsorption by V12O32-DNA@SVM composite and stripping with SDS gives rise to a clear band for S1 at 96 kDa (Lane 6), meanwhile the other bands are favorably eliminated. This well suggests a high purity for the recovered S1.

Figure 7. (A) SDS-PAGE results for the isolation of myosin S1 from porcine heart protein extract after cascade extraction. Lane 1: porcine heart protein extract from left ventricle; Lane 2: porcine heart protein extract after adsorption with V12O32-DNA@SVM composite as adsorbent; Lane 3: the collected myosin by stripping with 1% SDS solution; Lane 4: α-chymotryptic digest of the collected myosin; Lane 5: the α-chymotryptic digestion solution after adsorption with V12O32-DNA@SVM composite; Lane 6: the recovered myosin S1 by stripping with 1% SDS solution. (B) MALDI-TOF MS peptide map of the purified myosin S1 by tryptic digestion.


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The purified myosin S1 is afterwards subject to tryptic digestion procedure and identified by MALDI-TOF MS, and the peptides mass spectra acquired are shown in Figure 7B. The list of the mass peptides unambiguously identified myosin S1 from porcine heart by database searching using MASCOT server. Moreover, unique peptides of myosin S1 (observed mass at 1899.9 and 2384.0) are entirely detected. Table 1 provides further information for the identified myosin S1 isoform, including accession number, protein and gene names, domain position, matched peptide sequence and its observed mass and sequence coverage. In general, the identified myosin S1 heavy chain has a molecular weight of 96457 Da and pI 8.67 which is assigned to the myosin-2 isoform and originated from the MYH2_PIG gene. The amino acid sequence of S1 heavy chain is at the position 1-842 in myosin-2. The matched peptide sequences are corresponding to the observed mass from the peptides mass spectra. The obtained 38% of sequence coverage is obviously higher than that of the identification of myosin heavy chain isoforms by previous studies, where a LC-MS/MS approach resulted in a maximum sequence coverage of 29%47, and an improved technique of SDS-PAGE and 2DE (two dimensional electrophoresis) provided a maximum sequence coverage of 20%48.



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Table 1. Myosin S1 isoform and matched peptides identified of porcine heart by MALDI-TOF MS

Access. no.

Significant hits

Position

Matched peptide sequences

and observed mass

Q9TV63

Myosin-2

1-842

SSDQEMAIFGEAAPYLR(1899.9)

[MYH2_PIG]

[S1 heavy chain]

LQQFFNHHMFVLEQEEYK(2384.0)

Sequence

coverage (%)

38

CONCLUSIONS The present work demonstrated the conjugation of V12O32-DNA conjugate with streptavidin-coated magnetic nanoparticle (SVM) to obtain a novel composite, V12O32-DNA@SVM. The composite possesses favorable biocompatibility and exhibits high selectivity for the adsorption of myosin with a favorable adsorption capacity. It provides practical feasibility for the selective isolation of myosin subfragment S1 from the α-chymotryptic digest of purified porcine heart myosin by a cascade extraction procedure. The characterization of myosin S1 has implications for elucidating the mechanism of cardiac contraction and treating certain heart conditions. By taking advantage of the high-affinity binding of polyoxometalates (POMs) with the protein domain, for the first time, this report demonstrates the vast potential of the use of POMs as a promising adsorbent for the purification of specific proteins.


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EXPERIMENTAL PROCEDURES Materials and reagents. All chemicals used in the present study are at least of analytical reagent grade unless otherwise specified. Myosin (MYO), myohemoglobin (MYB), transferrin (TRF), cytochrome c (cyt-c), bovine serum albumin (BSA), N-tosyl-l-lysine chlorometyl ketone (TLCK)-treated α-chymotrypsin and tryspin are purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein molecular weight marker (Broad, 3597A) is obtained from Takara Biotechnology (Dalian, China). Tetrabutylammonium bromide (TBA), ethylene diamine tetraacetic aciddiethyl (EDTA), ether (Et2O), ethanol (Et2OH), dimethylsulfoxide (DMSO), glutaraldehyde (GA), hydrogen peroxide (30%, w/w), sodium-dodecyl sulphate (SDS), phenylmethylsulfonyl fluoride (PMSF), tris(hydroxymethyl)aminomethane (Tris), Coomassie brilliant blue G250, triethylamine, imidazole, acetone, acetonitrile, acrylic acid, hydrogen chloride, ethylene imine polymer, triethyl ammonium acetate, ethylene glycol, CHCl3, V2O5, VOSO4·3H2O, FeCl3·6H2O and SnCl2 are obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Other chemicals, including NaCl, KCl, H3PO4, H3BO3, HAc, NaAc, NaOH, KH2PO4, Na2HPO4 and sodium citrate are supplied by Bodi Chemical Holding Co. Ltd (Tianjin, China). HPLC purified synthetic oligonucleotide sequences are the product of Sangon (Shanghai). Deionized water (ddH2O) of 18 MΏ cm is used throughout. Instrumentations. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra are recorded by using a Nicolet-6700 FT-IR spectrophotometer (Thermo Fisher Scientific, USA) from 650 to 2000 cm−1 with ZnSe crystal. Raman



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spectra are recorded on an XploRA ONE laser Raman spectrometer (Horiba Scientific, Ltd., France). SEM images are obtained on a ZEISS Ultra/Plus scanning electron microscope (ZEISS, Germany). Circular dichroism (CD) spectra are obtained on a MOS-450 (Bio-Logic, France) automatic recording spectropolarimeter at 293 K. MALDI-TOF MS experiments are performed on an ultrafleXtreme time-of-flight mass spectrometer (Bruker Bremen, Germany) in the positive-ion reflector mode with a 337-nm nitrogen laser. The mass spectra are acquired with an available accelerating voltage of 20kV in a m/z range of 700-3500. U-3900 UV-vis spectrophotometer (Hitachi, Japan) with a 1-cm quartz cell is used for the quantitative detection of protein. A PB-10 pH Meter (Beijing Sartorius Instruments Co., Ltd., China) is used for pH monitoring. Ultrafiltration is performed on an Amicon utra-0.5 mL centrifugal filter of 3 kDa (Millipore Corporation, Bedford). Preparation of (TBA)4[V12O32{Sn(CH2)2CO2H}] (V12O32-COOH). (TBA)4[V12O32(CH3CN)] (shortly as V12O32)40 and Cl3Sn(CH2)2CO2H32 are first obtained by following previously reported procedures and the detailed experimental processes are described in the Supporting information. V12O32-COOH is thereafter prepared by following a literature approach with minor modifications16. Briefly, V12O32 (532.5 mg, 0.25 mmol) is dissolved with stirring in 10 mL acetonitrile and a brown solution is obtained. Afterwards, Cl3Sn(CH2)2CO2H (150 mg, 0.5 mmol) is added to the homogeneous solution. The resultant mixture is stirred vigorously at room temperature under argon atmosphere for 24 hours. After removal of the undissolved Cl3Sn(CH2)2CO2H by filtration, the dark green filtrate is collected in a


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25-mL conical flask and concentrated at 60°C under vacuum for overnight. The obtained insoluble product is dissolved in 5 mL acetone followed by addition of 10 mL of mixed solvent (EtOH/Et2O, 1/20) to produce a dark green precipitate, which is finally collected and dried at 60°C for 1 h. Preparation of V12O32-DNA conjugate. V12O32-DNA conjugate is prepared with the following procedure38. In a 1.0-mL centrifuge tube, 100 µL of 100 µmol L-1 oligonucleotide (5’-NH2-(CH2)6PO3-ATTACAATGGCAGGCTCCAGA-biotin-3’) is lyophilized and reconstituted in 10 µL ddH2O, followed by adding 5 µL of triethyl amine and 75 µL of 2.5 mM V12O32-COOH solution (57.2 mg V12O32-COOH is dissolved in 10 mL of DMSO). The mixture is maintained at 37°C for 30 h under continuous shaking. Afterwards, 10 µL of triethyl ammonium acetate and 500 µL of acetone are added and the mixture is further incubated for overnight at -20°C. The mixture is centrifuged at 13000 rpm for 10 min and the supernatant is discarded. The whitish pellet at the bottom of the centrifuge tube is then collected and washed twice with acetone to remove the un-reacted V12O32-COOH. After quick evaporation of acetone, 100 µL of ddH2O is added and the obtained V12O32-DNA solution is stored at -20°C. Preparation of the V12O32-DNA@SVM composite. Streptavidin-modified magnetic nanoparticles (SVM) are obtained according to a reported procedure49 and the detailed experimental processes are described in the Supporting information. SVM is well-dispersed in phosphate buffer (pH 8.0) by mild sonication for 5 min to obtain a black aqueous suspension (10 mg mL-1). Then V12O32-DNA@SVM composite is



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prepared with the following simple procedure. 100 µL of the SVM suspension is taken and washed twice with 15 mmol L-1 saline sodium citrate buffer (SSC). After discarding the supernatant, 100 µL of V12O32-DNA solution and 100 µL of 30 mmol L-1 SSC are introduced and incubated for 60 min at room temperature, to facilitate SVM to interact with the biotinylated V12O32-DNA. Finally, the supernatant is discarded and the obtained particles are washed twice with ddH2O for future use. Protein adsorption/desorption by V12O32-DNA@SVM. Myosin, transferrin, bovine serum albumin, cytochrome c and myoglobin are selected as protein models to investigate their adsorption behaviors on the V12O32-DNA@SVM composite. The acidity of protein solutions are controlled within pH 4-10 by dilute NaOH or HCl solutions. In general, 0.1 mL of protein solution is mixed with 0.1 mg of the V12O32-DNA@SVM composite and the mixture is shaken vigorously for 30 min to facilitate the adsorption of protein species. Afterwards, the mixture undergoes separation via a magnet and the supernatant is used for quantifying the residual protein content by monitoring the absorbance at 595 nm after binding with the Coomassie brilliant blue G250 (Bradford method). The adsorption efficiency is therefore calculated. V12O32-DNA@SVM composite is collected and pre-washed with 0.1 mL of imidazole (500 mmol L-1) to remove the nonspecifically adsorbed proteins. Thereafter, 0.1 mL of SDS solution (1%, m/m) is then added and the mixture is oscillated for 20 min to strip the adsorbed protein from the surface of V12O32-DNA@SVM composite.


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The supernatant is collected for evaluating the elution efficiency or for ensuing investigations. Pretreatment of porcine heart. Fresh porcine heart from a local market is thoroughly washed with ddH2O to remove blood. The left ventricle is cut down and chopped to fine powder. Small portions of the minced meat are packed into PET bags and stored at -20°C for future use. For protein extraction from the porcine heart, a portion of the frozen meat powder is repeatedly homogenized and washed with a solution containing 0.1mol L-1 of KCl and 20 mmol L-1 of Tris-HCl at pH 7.550. The myofibril suspension is then filtered through two layers of cotton gauze, and the filtrate is collected for the ensuing study of protein adsorption by the V12O32-DNA@SVM composite.

ASSOCIATED CONTENT *Supporting Information

AUTHOR INFORMATION *

Corresponding author.

E-mail address: [email protected] (X.W. Chen), [email protected] (J.H. Wang). Tel: +86 24 83688944; Fax: +86 24 83676698

Notes

The authors declare no competing financial interest.



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ACKNOWLEDGEMENTS The authors appreciate financial support from the Natural Science Foundation of China (21475017 and 21235001), Fundamental Research Funds for the Central Universities (N150502001, N140505003, N141008001, N160302001), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201702). Supporting Information Available: Preparation of TBA4[V10O26]; Preparation of TBA4[V12O32(CH3CN)]; Preparation of Cl3Sn(CH2)2COOH; Preparation of NH2-Fe3O4; Preparation of streptavidin-modified magnetic nanoparticles (SVM).


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Graphical Abstract


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Selective Isolation of Myosin Subfragment-1 with a DNA

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