Samples were cross-linked using the zero-length cross-linking reagent DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) and analyzed as described previously66 (link). Briefly, the cross-linked sample (approximately 50 µg protein) was digested with endoproteinase Lys-C (2.5 h, 1:100 enzyme-to-substrate ratio) and trypsin (overnight, 1:50). The digested sample was purified by solid-phase extraction and fractionated using peptide-level size-exclusion chromatography (SEC; Superdex Peptide, GE). Three fractions collected from the SEC experiment were analyzed in duplicate by LC–MS/MS on an Orbitrap Elite mass spectrometer (ThermoFisher Scientific) in data-dependent acquisition mode with 60 min gradients. MS/MS data was searched using xQuest67 (link) against a database containing the TRiC subunits and contaminant proteins, and identifications were filtered at a false discovery rate of 5% at the peptide pair level as determined by xProphet. Distances were extracted using PDB (Protein Data Bank) entry 6NRA as a structural model.
Superdex peptide
Manufactured by GE Healthcare
Sourced in United States
Superdex Peptide is a size exclusion chromatography medium used for the purification of peptides and small proteins. It provides efficient separation based on molecular size, enabling the isolation of target molecules from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200, by GE Healthcare (2 mentions)
Peptide calibration standard 2, by Bruker (1 mentions)
Biacore x100 evaluation software, by Cytiva (1 mentions)
Surfactant p20, by GE Healthcare (1 mentions)
Biacore x100 system, by Cytiva (1 mentions)
Bolt gel, by Thermo Fisher Scientific (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Ace enzyme, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
Captopril, by Merck Group (1 mentions)
Mtgase, by Ajinomoto (1 mentions)
Cmp6050 plant growth chamber, by Conviron (1 mentions)
1200 series hplc, by Agilent Technologies (1 mentions)
Mts 810, by MTS Systems (1 mentions)
Akta pure 25 system, by GE Healthcare (1 mentions)
8 protocols using superdex peptide
1
Cross-Linking Mass Spectrometry of TRiC Complex
2
Solid-Phase Synthesis of Soluble Peptides
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The soluble peptides were manually synthesized by Fmoc chemistry in the solid phase on a 30-µmol scale [22] (link). Briefly, the Fmoc-amino acids were activated with a 1∶2 solution of Oxyme and DIC. The active amino acids were incorporated into Rink amide resin with a substitution degree of 0.61. Fmoc deprotection was then performed using 25% 4-methylpiperidine. These steps were repeated until the synthesis of each peptide was complete. The peptides were deprotected and released from the resin by treatment with a solution of 9.4% trifluoroacetic acid, 2.4% water, and 0.1% triisopropylsilane. The peptides were precipitated with cold diisopropyl ether and purified by Superdex Peptide (30 pg) (GE Healthcare, USA). The peptides were obtained with at least 90% purity, as confirmed by mass spectrometry using MALDI-TOF-TOF Autoflex III equipment (Bruker Daltonics, USA). Instrument calibration was achieved using Peptide Calibration Standard II (Bruker Daltonics, USA) as a reference, and a-cyano-4-hydroxycinnamic acid was used as the matrix.
3
Surface Plasmon Resonance of Heparin-PNA-Fc Binding
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Surface plasmon resonance (SPR) experiment was performed with the Biacore X100 system and Biacore X100 evaluation software (Biacore AB, Uppsala, Sweden). Heparin was coupled with biotin-hydrazide (Dojindo, Kumamoto, Japan) by reductive amination. Briefly, 1 mg of heparin solubilized in 125 μl of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5, was mixed with 250 μl of 72 mM biotin-hydrazide in dimethyl sulfoxide and 250 μl of 1 M sodium cyanoborohydride in 0.1 M MES, pH 5.5. After the mixture was allowed to stand at 20°C for 12 h, biotinylated heparin was purified on a column of Superdex peptide (3 × 250 mm, GE Healthcare). Biotinylated heparin was immobilized to streptavidin-coupled sensor chip SA (Biacore) until the resonance units reached approximately saturation (approximately 130 resonance unit (RU)). The binding of mutated PNA-Fc to heparin was measured in 10 mM HEPES, pH 7.4, containing 150 mM NaCl and 0.005% surfactant P20 (GE Healthcare) at 25°C with a flow rate of 20 μl/min. The mutated PNA-Fc at 1, 0.2, 0.04, 0.008, and 0.0016 μM was applied to the sensor chip. The regeneration of the chip surface was carried out with 50 mM glycine-HCl, pH 3.0, containing 150 mM NaCl and 0.005% surfactant P20.
4
Tetanus Toxin Fragment C Conjugation
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100 pmol tetanus toxin fragment C (TTC) is mixed with 4 nmol maleimide–DNA (complementary to the DNA in the PSNA corona) in 30 μL PBS pH 7.4 containing 500 μM TCEP. The reaction is left overnight at 4 °C in an orbital shaker. Conjugates are analyzed by polyacrylamide gel electrophoresis using a Bolt gel (Invitrogen) run at 200 V for 32 minutes in MES buffer (Invitrogen) and stained with Sypro Ruby (Invitrogen) following manufacturer's instructions (ESI Fig. 1a† ). TTC–DNA conjugates are purified using size-exclusion liquid chromatography (SE-LC) with a 10/300 column pre-packed with Superdex Peptide (GE) (ESI Fig. 1b† ). Conjugates are injected using a 0.5 mL loop. The column is eluted with 1.5 column volumes of PBS at 0.5 mL min−1. Conjugates elute between minutes 16 and 20. Purified conjugates are concentrated 3× using an Amicon Ultra 0.5 mL 30 kDa centrifugal filter (Millipore).
5
Enzyme-Assisted Oyster Protein Hydrolysis
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Oysters were purchased from a local market in October 2011 (Tongyeong, Korea). ACE enzyme (EC 3.4.15.1, Sigma A6778), pepsin (EC 3.4.23.1, 570 units/mg, Sigma P7125), trypsin (EC 3.4.21.4, 12,800 units/mg, Sigma T1426), N-Hisppuryl-His-Leu hydrate (HHL, Sigma H1635), and Captopril (Sigma C4042) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Several specific peptides (TAY, VK, KY, FYN, and YA) were synthesized at GL Biochem Ltd. (Shanghai, China). Sardine hydrolysate was purchased from Chosunmuyak Co. (Seoul, Korea). Alcalase 2.4 L (2.4 AU/g, endopeptidase, Bacillus licheniformis), Flavourzyme 500 MG (500 LAPG/g, endoprotease and exopeptidase, Aspergillus oryzae), Protamex 1.5 MG (1.5 AU/g, Bacillus protease, complex), and Neutrase 0.8 L (0.8 AU/g, endoprotease, Bacillus amyloliquefaciens) were obtained from Biosis Co. (Busan, Korea). MTGase (103 U/g) was obtained from Ajinomoto Co. (Tokyo, Japan). Acetonitrile and methanol were high-performance liquid chromatography (HPLC) grade. All other reagents were reagent grade. HiLoad Q-Sepharose, Superdex peptide, and Source 5RPC ST columns were purchased from GE Healthcare (Parsippany, NJ, USA). A Bondclone C18 column was purchased from Daiso Chemical Co. (Tokyo, Japan).
6
Thermal Hydrolysis of Sustainable Raw Materials
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Thermal hydrolysis of SRM was performed using a 5.5 L high temperature and high pressure stainless steel Parr reactor vessel (Parr 4582) equipped with Parr reactor controller (Parr 4848, Parr Instrument Company, Moline, IL, USA). Size exclusion high performance liquid chromatography (SEC-HPLC) analysis of hydrolysed protein was conducted using an Agilent Technologies 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler, variable wavelength UV detector, and two size exclusion columns – Superdex™ 200 and Superdex™ peptide (GE Healthcare Biosciences AB, Uppsala, Sweden). Viscosity of adhesive formulations was measured using an OFITE model 900 viscometer (OFI Testing Equipment, Houston, TX, USA). Hot pressing of plywood specimens was performed using a Carver hot press (Model 3890 Auto ‘M’, Carver Inc., Wabash, IN, USA). A CMP 6050 plant growth chamber (Conviron, Winnipeg, MB, Canada) was used for conditioning of plywood specimens prior to testing. The plywood specimens were tested using mechanical test system (MTS 810, MTS Systems Corporation, Eden Prairie, MN, USA) equipped with a 10 kN load cell. The scanning electron microscope (SEM) images of wood specimens were acquired using Zeiss Sigma 300 VP Field Emission SEM (Carl Zeiss AG, Oberkochen, Germany) operating at 20 kV.
7
Cross-linking Mass Spectrometry of Puf6:60S Complex
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Reconstituted Puf6:60S complexes were analyzed by XL-MS using chemical cross-linking with disuccinimidyl suberate (DSS)72 (link),73 . In total, 100 μM purified 60S ribosome was incubated with 200 μM Puf6 at 20 °C for 10 min in 60S binding buffer (150 mM NaCl, 20 mM HEPES pH7.5, 5 mM MgCl2, 5 mM β-mercaptoethanol) and cross-linked with DSS-d0/d12 (25 mM stock solution in anhydrous dimethylformamide; Creative Molecules) to a final concentration of 1 mM. The reaction was incubated at 37 °C for 30 min and subsequently quenched using 50 mM ammonium bicarbonate. Cross-linked complexes were reduced, alkylated, and digested sequentially with endoproteinase Lys-C and trypsin72 (link),73 . The digest was fractionated with size exclusion chromatography (Superdex Peptide, GE) and fractions were analyzed by LC-MS/MS on an Orbitrap Elite mass spectrometer72 (link). Data analysis was performed using xQuest/xProphet (v2.1.3) and results were filtered to a false discovery rate of <5% at the cross-linked peptide pair level74 (link). The identifications are summarized in Supplementary Data 2 , and the mass spectrometry data is deposited to the ProteomeXchange Consortium via the PRIDE69 (link) partner repository with the identifier PXD024131 .
8
Optimized C. vulgaris Hydrolysate MW Analysis
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The optimized C. vulgaris hydrolysate molecular weight (MW) distribution was determined using the AKTA Pure 25 system (GE Healthcare Life Sciences, Freiburg, Germany) coupled with two gel filtration columns (Superdex 200 increase10/300 G L and Superdex peptide, 10/300 G L) and controlled by UNICORN 7.0 software (GE Healthcare Life Sciences, Freiburg, Germany), as described previously [45 (link)]. A solution of 25 mM phosphate buffer (pH 7.0), 150 mM sodium chloride, and 0.2 g/L sodium azide was used as the mobile phase. An eluent flow of 0.5 mL/min and an absorbance of 280 nm were used to build samples and standard chromatograms.
To set up a standard curve, the following proteins and peptides, with known MW, were used: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa), and the antihypertensive peptide KGYGGVSLPEW (1.2 kDa). The hidden peak analysis was performed using software OriginPro 2021 v9.8.0.200.
To set up a standard curve, the following proteins and peptides, with known MW, were used: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa), and the antihypertensive peptide KGYGGVSLPEW (1.2 kDa). The hidden peak analysis was performed using software OriginPro 2021 v9.8.0.200.
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