Between 2 and 10% of the collected fractions were analysed by LC–MS/MS using a nanoAcquity UPLC system (Waters Corporation, Manchester, UK) connected online to an LTQ-Orbitrap Velos Pro instrument (Thermo). Peptides were separated on a BEH300 C18 (75 μm × 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters) using a stepwise 60 min gradient between 3 and 85% (v/v) ACN in 0.1% (v/v) FA. Data acquisition was performed using a TOP-20 strategy where survey MS scans (m/z range 375–1,600) were acquired in the Orbitrap (R=30,000) and up to 20 of the most abundant ions per full scan were fragmented by collision-induced dissociation (normalized collision energy=40, activation Q=0.250) and analysed in the LTQ Orbitrap. To focus the acquisition on larger cross-linked peptides, charge states 1, 2 and unknown were rejected. Dynamic exclusion was enabled with repeat count=1, exclusion duration=60 s, list size=500 and mass window ±15 p.p.m. Ion target values were 1,000,000 (or 500 ms maximum fill time) for full scans and 10,000 (or 50 ms maximum fill time) for MS/MS scans. All the samples were analysed in at least technical duplicates.
Beh300 c18
Manufactured by Waters Corporation
Sourced in United States
The BEH300 C18 is a chromatography column designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. It features a 1.7 μm particle size and a 300 Å pore size, which provide efficient separation and high-resolution analysis of a wide range of analytes.
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Lab products found in correlation
5 protocols using beh300 c18
1
Cross-Linked Peptides Analyzed by LC-MS/MS
2
Tryptic Peptide Separation and Identification
3
Peptide Separation and Analysis by nanoUPLC-MS/MS
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Peptides were separated with a BEH300 C18 (75 μm x 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters) using a stepwise 145 min gradient from 3% to 85% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 300 nl/min. The LTQ-Orbitrap Velos Pro instrument was operated in data-dependent mode. Parameters for the CID-based method used one survey MS scan acquired in the orbitrap followed by up to 20 fragmentation scans (TOP20) of the most abundant ions analysed in the LTQ. Only charge states of two and higher were allowed for fragmentation. Essential MS settings were: full MS: AGC = 106, maximum ion time = 500 ms, m/z range = 375–1600, resolution = 30 000 FWHM; MS2: AGC = 30 000, maximum ion time = 50 ms, minimum signal threshold = 1500, dynamic exclusion time = 30 s, isolation width = 2 Da, normalized collision energy = 40, activation Q = 0.25.
4
High-throughput Peptide Separation and Analysis
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The 10 fractions obtained by high pH fractionation were analyzed using a nanoAcquity UPLC system (Waters GmbH) connected online to a LTQ-Orbitrap Velos Pro instrument (Thermo Fisher Scientific GmbH). Peptides were separated on a BEH300 C18 (75 μm × 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters GmbH) using a stepwise 145 min gradient between 3% and 85% (vol/vol) ACN in 0.1% (vol/vol) FA. Data acquisition was performed using a TOP-20 strategy where survey MS scans (m/z range 375–1,600) were acquired in the Orbitrap (R = 30,000 FWHM) and up to 20 of the most abundant ions per full scan were fragmented by collision-induced dissociation (normalized collision energy = 35, activation Q = 0.250) and analyzed in the LTQ. Ion target values were 1 × 106 (or 500 ms maximum fill time) for full scans and 1 × 105 (or 50 ms maximum fill time) for MS/MS scans. Charge states 1 and unknown were rejected. Dynamic exclusion was enabled with repeat count = 1, exclusion duration = 60 s, list size = 500 and mass window ±15 ppm.
5
Peptide Identification via ESI-TOF-MS
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ESI-TOF-MS was used for the identification of peptides from HPLC Fractions as described by Dang et al. (15 (link)). Electrospray ionization (ESI) was used in positive ion mode, source temperature was set at 150°C, desolvation temperature was set at 550°C, scan range was m/z 50–1200. The most active fraction (F6) was resuspended in water at a concentration of 1 mg/mL. The filtrate was then analyzed with the ESI-TOF mass spectrometer of the ACQUITY UPLC H-Class HPLC system (Waters, USA). The column used was BEH300 C18 (2.1 × 100 mm; 1.7 μm) (Waters, USA), the flow rate was 0.2 mL/min, and the injection volume was 10 μL. All MS and MS/MS spectra were collected and analyzed using Mass Lynx (Waters version 4.1). The PepSeq program of Biolynx (Waters Corp.) software was used for sequencing. These peptides were custom-synthesized by Sangon Biotech Co., Ltd., (Shanghai, China) (16 ).
The purity of these peptides was above 99%.
The purity of these peptides was above 99%.
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