Proswift rp 4h column

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ProSwift RP-4H column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a durable polymeric packing material that provides high chemical and mechanical stability. The column offers efficient performance for various applications, including the analysis of small molecules, peptides, and proteins.

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ProSwift™ RP-4H 1 ×50 mm column ProSwift RP-4H monolithic column ProSwift RP-4H

11 protocols using proswift rp 4h column

Comprehensive Biophysical Characterization of Biomolecules

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UV–vis
absorption measurements were conducted on a Cary UV–vis 100
spectrophotometer (Agilent, USA). Protein concentration was determined
by UV–vis analysis on a Nanodrop 1000 instrument (Nanodrop,
USA) by monitoring absorbance at 280 nm. Electrospray LC/MS analysis
of proteins and their bioconjugates was performed using an Agilent
1200 series liquid chromatograph (Agilent Technologies, USA) that
was connected in-line with an Agilent 6224 time-of-flight (TOF) LC/MS
system equipped with a Turbospray ion source. Protein samples were
run with a Proswift RP-4H column (Dionex, USA). Protein mass reconstruction
was performed on the charge ladder with Mass Hunter software (Agilent,
USA). High-performance liquid chromatography (HPLC) was performed
on Agilent 1200 Series HPLC Systems (Agilent, USA). Sample analysis
for all HPLC experiments was achieved with an in-line diode array
detector and in-line fluorescence detector. SEC was performed using
a Polysep-GFC-P-5000 column (4.6 × 250 mm) (Phenomenex, USA)
at 1.0 mL/min using a mobile phase of 10 mM sodium phosphate buffer,
pH 7.2. DLS was performed on a Zetasizer Nano Series (Malvern Instruments,
UK). Measurements were taken in triplicate at protein concentrations
of 0.2–1.0 mg/mL in 10 mM sodium phosphate buffer, pH 7.2,
at 25 °C.

Bischoff A.J., Hamerlynck L.M., Li A.J., Roberts T.D., Ginsberg N.S, & Francis M.B. (2023). Protein-Based Model for Energy Transfer between Photosynthetic Light-Harvesting Complexes Is Constructed Using a Direct Protein–Protein Conjugation Strategy. Journal of the American Chemical Society, 145(29), 15827-15837.

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Protein Characterization by LC-MS

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The chemical protein modification was monitored using LC-MS. LC-MS was performed using a Micromass LCT (ESI-TOF-MS) and a Shimadzu Prominence HPLC equipped with a Dionex Proswift RP-4H column. The column was run at 0.4 ml min⁻¹. Solvents used were water with 0.1% formic acid and 95:5 Acetonitrile: water with 0.1% formic acid. The proteins were separated using a gradient from 5% B to 95% B in 4 min. After this the column was washed for 2 min with 95% B and then equilibrated with 5% B for 2.5 min. The electrospray source was set to 3200 V capillary voltage and 25 V cone voltage.
Data were analysed using Masslynx and calibrated using myoglobin. Deconvolution was performed using the maximum entropy algorithm.

Lercher L., Raj R., Patel N.A., Price J., Mohammed S., Robinson C.V., Schofield C.J, & Davis B.G. (2015). Generation of a synthetic GlcNAcylated nucleosome reveals regulation of stability by H2A-Thr101 GlcNAcylation. Nature Communications, 6, 7978.

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Mass Spectrometry Characterization of Ec1-LoPE

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Ec1-LoPE (Fig. 1) was produced and characterized as described previously (38 (link)). To confirm the identity, the molecular weight of the protein was determined using electrospray ionization mass spectrometry (Impact II instrument, Bruker Corp.). The spectrometer worked on line with Dionex UltiMate 3000 ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Inc.) equipped with a ProSwift RP-4H column (1×50 mm, Thermo Fisher Scientific, Inc.). The chromatographic system used two solvents: Solvent A (3% acetonitrile, 0.1% formic acid in water) and solvent B (95% acetonitrile, 0.1% formic acid in water), and the flow rate was 200 µl/min. The following gradient profile was used: 4% solvent B for 2 min, 4–90% solvent B within 6 min, 90% solvent B for 2 min, 90–4% solvent B within 1 min followed by 4% solvent B for 4 min. The found molecular weight (43,061 kDa) was in excellent agreement with the calculated molecular weight (43,061 kDa) (Fig. 2A). According to HPLC analysis, the purity of Ec1-LoPE was >99% (Fig. 2B).

Xu T., Liu Y., Schulga A., Konovalova E., Deyev S.M., Tolmachev V, & Vorobyeva A. (2022). Epithelial cell adhesion molecule-targeting designed ankyrin repeat protein-toxin fusion Ec1-LoPE exhibits potent cytotoxic action in prostate cancer cells. Oncology Reports, 47(5), 94.

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Intact Protein Characterization by EThcD MS

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Intact trimers were diluted in solvent A (97.5% water, 2.5% ACN and 0.1% formic acid) to 0.03 mg/mL The chromatography was performed using an Ultimate 3000 ultra-high performance liquid chromatograph (ThermoFisher Scientific, San Jose, CA) interfaced to an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific). Three μL of intact sample were injected, concentrated, and desalted on a PepSwift Monolith trap (200 μm × 5 mm) for 5 mins before separation on a ProSwift RP-4H column (100 μm × 25 cm) (ThermoFisher Scientific) with a gradient of 20% to 40% solvent B (75% ACN, 25% water, 0.1% formic acid) over 15 mins. The potential for in-source fragmentation was set to 10V. Precursor and fragment ion masses were acquired with a resolution of 120,000. Fragmentation was triggered in data dependent mode by electron transfer supported by chemical ionization (EThcD) with a 6 msec ETD reaction time and supplemental activation at 10% normalized HCD.

Lee A.E., Geis-Asteggiante L., Dixon E.K., Kim Y., Kashyap T.R., Wang Y., Fushman D, & Fenselau C. (2016). Preparing to Read the Ubiquitin Code: Characterization of Ubiquitin trimers by Top-down Mass Spectrometry. Journal of mass spectrometry : JMS, 51(4), 315-321.

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Nano-LC-MS/MS Protein Separation

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Resuspended protein fractions (6 μL) were injected onto a PepSwift RP-4H trap column (150 μm ID × 2 cm). After loading the sample at 10 uL/min, the trap was switched in-line with a Thermo ProSwift RP-4H column (100 μm ID × 50 cm) for protein separation. Mobile phases were delivered using a Dionex Ultimate 3000 RSLCnano system. Proteins were eluted from the ProSwift RP-4H column with a mobile phase flow rate of 1 μL/min. Solvent A was described above. Solvent B was 5% water, 95% acetonitrile, 0.2% formic acid. A linear gradient was used with slope change points: 5% B at 0 min.; 15% B at 5 min.; 55% B at 80 min.; 95% B hold from 83 to 102 min. Eluent was directed to a 15 μm nanoelectrospray tip (New Objective, Waltham, MA) held at 1.9–2.1 kV. Mass spectrometry measurements were performed on an Orbitrap Elite (Thermo Scientific, Bremen, Germany) mass spectrometer operating in “protein mode” and fitted with a custom nanoelectrospray ionization source. A top-2 data-dependent acquisition strategy was employed as described previously using higher-energy collisional dissociation (HCD).^{17 (link)} Biological and technical replicates were randomized across the study to minimize bias.

Davis R.G., Park H.M., Kim K., Greer J.B., Fellers R.T., LeDuc R.D., Romanova E.V., Rubakhin S.S., Zombeck J.A., Wu C., Yau P.M., Gao P., van Nispen A.J., Patrie S.M., Thomas P.M., Sweedler J.V., Rhodes J.S, & Kelleher N.L. (2018). Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains. Analytical chemistry, 90(6), 3802-3810.

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Heterodimer Protein Characterization by LC-MS

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The samples were deglycosylated using PNGase F prior to the measurement. To determine the correct pairing of the heterodimer chains, the proteins were directly injected to a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) system (LC: Dionex Ultimate 3000 LC). A gradient from 20 to 80% acetonitrile in 0.05% trifluoroacetic acid (using a Thermo ProSwift™ RP-4H column of 0.2 x 250 mm dimensions) at a flow rate of 8 μL/min was applied (30 min gradient time). Detection was performed with a Q-TOF instrument (Bruker maXis 4G) equipped with the standard ESI source in positive ion, MS mode (range: 750–5000 Da). Instrument calibration was performed using ESI calibration mixture (Agilent). Data was processed using Data Analysis 4.0 (Bruker) and the spectrum was deconvoluted by MaxEnt.

Wozniak-Knopp G., Stadlmayr G., Perthold J.W., Stadlbauer K., Gotsmy M., Becker S, & Rüker F. (2018). An antibody with Fab-constant domains exchanged for a pair of CH3 domains. PLoS ONE, 13(4), e0195442.

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Protein Detection via HPLC-MS

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Protein samples (0.1 mg/ml, 1 µl) were injected with an UltiMate3000® HPLC system (UHPLC⁺ focused, Dionex) into a ProSwift™ RP-4H column (1× 50 mm, Thermo) at a flow rate of 0.7 ml/min. The proteins eluted with a linear gradient of 5–100% acetonitrile (0.1% formic acid) in 6 min. The desalted samples were ionized and analyzed by a LCQ fleet^™ system (Thermo) combining electro spray ionization with an ion trap mass analyzer.

Vieweg S., Mulholland K., Bräuning B., Kachariya N., Lai Y.C., Toth R., Singh P.K., Volpi I., Sattler M., Groll M., Itzen A, & Muqit M.M. (2020). PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72. Biochemical Journal, 477(9), 1651-1668.

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Intact Protein Tetramers Mass Spectrometry

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Intact tetramers were diluted to 0.03 mg/mL in Solvent A (97.5% water, 2.5% ACN and 0.1% formic acid). The chromatography was performed using an Ultimate 3000 ultra-high performance liquid chromatograph (ThermoFisher Scientific, San Jose, CA) interfaced to a orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific). Five µL (~4.4 pmol) was injected, concentrated and desalted on a PepSwift Monolith trap (200 µm × 5 mm) for 5 min at 99% Solvent A before separation on a ProSwift RP-4H column (100 µm × 25 cm) (ThermoFisher Scientific) with a gradient of 30% to 50% solvent B (75% ACN, 25% water, 0.1% formic acid) over 15 min. Precursor and fragment ion masses were acquired with a resolution of 120,000 at m/z 200 using “intact protein mode” with 1mtorr ion routing multipole pressure. Data dependent MS/MS was carried in top-N mode (N=2) with a precursor list of m/z values calculated for each tetramer. Isolated parents ions were fragmented using electron transfer dissociation supplemented with collisionally induced dissociation (ETciD) with a 3 msec ETD reaction time and supplemental activation at 10% normalized CID and averaging 20 microscans.

Lee A.E., Geis-Asteggiante L., Dixon E.K., Miller M., Wang Y., Fushman D, & Fenselau C. (2016). Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers. Journal of mass spectrometry : JMS, 51(8), 629-637.

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Peptide and Intact Protein Separation

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Peptide samples were separated using a Vanquish Horizon UHPLC system coupled with a Hypersil GOLD C18 column (1.9 μm, 175 Å, 2.1 × 200 mm), maintained at 60 °C, and connected to an Ion Max API source (Thermo Fisher Scientific, Waltham MA). A binary gradient of solvent A (0.1% formic acid in water) and solvent B (10% water, 10% isopropanol, 0.1% formic acid in acetonitrile) was used, increasing from 5 to 40% solvent B over 60 min at a flow rate of 60 μL/min. Intact protein samples were separated using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham MA) coupled with a ProSwift RP-4H column (1 × 250 mm), maintained at 60 °C, and connected to an Ion Max API source. A binary gradient of solvent A (0.2% formic acid in water) and solvent B (0.2% formic acid in acetonitrile) was used, increasing from 2 to 50% solvent B at a flow rate of 200 μL/min. Acquisition times for intact protein chromatography ranged from five minutes to two hours.

Neil J.R., Verma A., Kronewitter S.R., McGee W.M., Mullen C., Viirtola M., Kotovuori A., Friedrich H., Finell J., Rannisto J., Syka J.E, & Stephenson JL J.r. (2021). Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein. Scientific Reports, 11, 18309.

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Cation Exchange Resin LC-MS Analysis

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For MS analysis, samples were directly injected into a liquid chromatography electrospray ionization MS (LC-ESI-MS) system (Dionex Ultimate 3000 LC, Thermo Fisher Scientific Inc., USA). We injected 5 μL of Source 15S (GE Healthcare Life Sciences, USA) cation exchange resin eluate via μL-pick up using 0.1% formic acid (Acros Organics, Belgium) as the transport liquid to fill up the 20 μL sample loop. In the case of the KanCap G (Kaneka Corp., Japan) fraction, a full loop mode injection was performed. For sample preparation, a 3D chromatography approach was used, as described in detail in [33] . Samples were run on a ProSwift RP-4H column (0.2 x 250 mm; Thermo Fisher Scientific Inc.) operated with a 20-80% acetonitrile (VWR®, USA) gradient in 0.05% trifluoroacetic acid (Thermo Fisher Scientific) at a flow rate of 8 μL/min and a gradient time of 30 min. Detection was performed via the maXis 4G quadrupole time-of-flight (Q-TOF) instrument (Bruker Corp., USA) equipped with the ESI source in positive ion MS mode with a range of 400-3000 Da. For calibration, an ESI calibration mixture (Agilent Technologies, USA) was used. Data processing was performed using Data Analysis 4.0 (Bruker). MaxEnt was used for spectrum deconvolution in the mass range of 10,000-100,000 Da.

Fink M., Schimek C., Cserjan‐Puschmann M., Reinisch D., Brocard C., Hahn R., & Striedner G. (2020). Combinatorial analysis of host-leader-Fab can greatly improve integrated process design.

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