Beh c18 particles

Manufactured by Waters Corporation

The BEH C18 particles are a type of chromatographic stationary phase used in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. They are made of bridged ethylene hybrid (BEH) material and feature a C18 alkyl chain bonded to the surface, providing a nonpolar stationary phase for the separation of a wide range of analytes.

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BEH C18 (162 citations)

9 protocols using beh c18 particles

Peptide Identification by LC-MS/MS

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Peptides in each fraction were reconstituted in 0.1% FA, 3% ACN and subjected to reversed phase LC-MS/MS analysis with a Q-Exactive HF orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) interfaced with a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded onto a 75 μm i.d. microcapillary column custom-packed with 15 cm of BEH C18 particles (1.7 μm, 130 Å, Waters). Peptides were separated with a 90 min gradient from 3% to 30% ACN with 0.1% FA, followed by 10 min to 75% ACN and then 10 min to 95% ACN. After that, the column was re-equilibrated with 3% ACN for 15 min to prepare for the next injection.
The mass spectrometer was operated in a top 15 data-dependent acquisition mode. Survey scans of peptide precursors from m/z 300 to 2000 were performed at a resolving power of 60K and an AGC target of 2×10⁵ with a maximum injection time of 150 ms. The top 15 intense precursor ions were selected and subjected to the stepped HCD fragmentation at normalized collision energy of 22, 30, and 38% followed by tandem MS acquisition at a resolving power of 30K and an AGC target of 5×10⁴, with a maximum injection time of 250 ms. Precursors were subjected to a dynamic exclusion of 15s with a 10 ppm mass tolerance.

Cui Y., Yang K., Tabang D.N., Huang J., Tang W, & Li L. (2019). Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-glyco and Phospho- Peptides. Journal of the American Society for Mass Spectrometry, 30(12), 2491-2501.

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Shotgun Proteomic Analysis Pipeline

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Approximately 0.6 μg protein digest, estimated by BCA protein assay, was injected into a Waters nanoAcquity HPLC coupled to an ESI ion-trap/Orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific, Waltham, MA). Peptides were separated on a 100 μm inner diameter column packed with 20 cm of 3 μm MAGIC aqC18 beads (Bruker-Michrom, Auburn, CA), and eluted at 0.3 μL/min in 0.1% FA with a gradient of increasing ACN over 2.5 h. As noted in the Results section, certain samples were separated on a column packed with 20 cm of 1.7 μm BEH C18 particles (Waters, Milford, MA), and a heater cartridge was used to keep the capillary column at 60 °C. A full-mass scan (300-1500 m/z) was performed in the Orbitrap at a resolution of 60,000. The ten most intense peaks were selected for fragmentation by higher-energy collisional dissociation (HCD) at 42% collision energy, then analyzed with a resolution of 7500 and an isolation width of 2.5 m/z. Dynamic exclusion was enabled with a repeat count of 1 over 30 s and an exclusion duration of 120 s. All MS raw files may be downloaded from the PeptideAtlas data repository [42 (link)] by the following link: http://www.peptideatlas.org/PASS/PASS00557.

Li Q., Uygun B.E., Geerts S., Ozer S., Scalf M., Gilpin S.E., Ott H.C., Yarmush M.L., Smith L.M., Welham N.V, & Frey B.L. (2015). Proteomic Analysis of Naturally-Sourced Biological Scaffolds. Biomaterials, 75, 37-46.

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Ultra-high Pressure Capillary Column Packing

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A laser puller (Sutter Instruments Co., Novato, CA) was used to generate electrospray emitter tips (~10 × 25 μm inner-outer diameter) on 75 ID × 360 μm OD bare-fused silica capillary columns (Polymicro Phoenix, AZ). The tips were briefly etched with 100% hydrofluoric acid and plugged with 5 μm, 130 Å pore size Bridged Ethylene Hybrid (BEH) C18 particles (Waters, Milford, MA) using an in-house made pressure injection cell with maximum gas pressure rating of ~1500 psi. Then, the column was packed with 1.7 μm diameter, 130 Å pore size BEH C18 particles (Waters, Milford, MA) using the ultra-high pressure (uHP) column packing station, reaching a maximum pressure of ~20,000 to ~30,000 psi.^{16 (link)}

Zhang Z., Dubiak K.M., Shishkova E., Huber P.W., Coon J.J, & Dovichi N.J. (2022). High throughput, comprehensive single cell proteomics analysis of Xenopus laevis embryos at 50-cell stage using a microplate-based MICRO-FASP system. Analytical chemistry, 94(7), 3254-3259.

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Peptide Separation and Identification by Nano-HPLC-MS

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Reconstituted peptide solution was injected into a Waters nanoAcquity HPLC coupled to an ESI ion-trap/Orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific, Waltham, MA). Peptides were separated on a 100 μm inner diameter column packed with 20 cm of 1.7 μm BEH C18 particles (Waters, Milford, MA), and eluted at 0.3 μL/min in 0.1% FA with a gradient of increasing ACN over 2.5 h. A heater cartridge was used to keep the capillary column at 60 °C. A full-mass scan (300–1500 m/z) was performe d in the Orbitrap at a resolution of 60,000. The ten most intense peaks were selected for fragmentation by higher-energy collisional dissociation (HCD) at 42% collision energy, then analyzed with a resolution of 7,500 and an isolation width of 2.5 m/z. Dynamic exclusion was enabled with a repeat count of 1 over 30 s and an exclusion duration of 120 s.

Gilpin S.E., Li Q., Evangelista-Leite D., Ren X., Reinhardt D.P., Frey B.L, & Ott H.C. (2017). Fibrillin-2 and Tenascin-C Bridge the Age Gap in Lung Epithelial Regeneration. Biomaterials, 140, 212-219.

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Shotgun Proteomics Workflow

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Each fraction was resuspended in 0.2% formic acid. 1 μg of peptide was loaded onto a 75 μm inner diameter × 360 μm outer diameter column (New Objective) packed in-house 1 to 30 cm with 1.7 micron BEH C18 particles (Waters Corp). The peptides were chromatographically separated over a 120-min reverse phase gradient (0.2% formic acid mobile phase A, 0.2% formic acid/80% ACN mobile phase B) using a Dionex UltiMate 3000 nano-HPLC (Thermo) at a column temperature of 50 °C and a flow rate of 335 nL/min. Eluting peptides were sprayed into an Orbitrap Eclipse using a Nanospray Flex ionization source (Thermo) at 2 kV. MS1 scans were recorded in the Orbitrap using a resolving power of 60,000, requiring an AGC target of 1e6 or a maximum injection time of 50 ms. MS1 scans included precursor ions ranging from 300-1350 m/z. The most intense ions were selected for HCD fragmentation (normalized collision energy 35%) and subsequent MS2 analysis until the 1 s duty cycle lapsed. Dynamic exclusion was set to 40 s and the MS2 isolation width was set to 1.5 Th. Only precursors of charge states 2–6 were selected. MS2 spectra were analyzed in the Orbitrap using a resolving power of 60,000 requiring an AGC target of 1e5 or a maximum inject time of 118 ms. MS2 scans included fragment ions ranging from 100 to 2000 m/z.

Niemi N.M., Serrano L.R., Muehlbauer L.K., Balnis C.E., Wei L., Smith A.J., Kozul K.L., Forny M., Connor O.M., Rashan E.H., Shishkova E., Schueler K.L., Keller M.P., Attie A.D., Friedman J.R., Pagan J.K., Coon J.J, & Pagliarini D.J. (2023). PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy. Nature Communications, 14, 6431.

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Optimized nano-LC-MS/MS protocol for peptide analysis

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Each fraction was resuspended in 0.2% formic acid. 1 μg of peptide was loaded onto a 75 μm inner diameter × 360 μm outer diameter column (New Objective) packed in-house 1 to 30 cm with 1.7 micron BEH C18 particles (Waters Corp). The peptides were chromatographically separated over a 120-minute reverse phase gradient (0.2% formic acid mobile phase A, 0.2% formic acid/80% ACN mobile phase B) using a Dionex UltiMate 3000 nano-HPLC (Thermo) at a column temperature of 50°C and a flow rate of 335 nL/min. Eluting peptides were sprayed into an Orbitrap Eclipse using a Nanospray Flex ionization source (Thermo) at 2 kV. MS1 scans were recorded in the Orbitrap using a resolving power of 60,000, requiring an AGC target of 1e6 or a maximum injection time of 50 ms. MS1 scans included precursor ions ranging from 300–1350 m/z. The most intense ions were selected for HCD fragmentation (normalized collision energy 35%) and subsequent MS2 analysis until the 1 second duty cycle lapsed. Dynamic exclusion was set to 40 seconds and the MS2 isolation width was set to 1.5 Th. Only precursors of charge states 2–6 were selected. MS2 spectra were analyzed in the Orbitrap using a resolving power of 60,000 requiring an AGC target of 1e5 or a maximum inject time of 118 ms. MS2 scans included fragment ions ranging from 100– 2000 m/z.

Niemi N.M., Serrano L.R., Muehlbauer L.K., Balnis C., Kozul K.L., Rashan E.H., Shishkova E., Schueler K.L., Keller M.P., Attie A.D., Pagan J., Coon J.J, & Pagliarini D.J. (2023). Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy. bioRxiv.

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Reverse Phase Peptide Separation and Orbitrap Mass Spectrometry

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Each sample was resuspended in 0.2% formic acid. 1.5 μg of peptide was loaded onto a 75 um inner diameter × 360 um outer diameter column (CoAnn Technologies) packed in-house1 to 30 cm with 1.7 micron BEH C18 particles (Waters Corp). The peptides were chromatographically separated over a 90-minute reverse phase gradient (0.2% formic acid mobile phase A, 0.2% formic acid/80% ACN mobile phase B) using a Vanquish Neo UHPLC (Thermo) at a column temperature of 50°C and a flow rate of 320 nL/minute. Eluting peptides were sprayed into an Orbitrap Eclipse using a Nanospray Flex ionization source (Thermo) at 2.2 kV. MS1 scans were recorded in the Orbitrap using a resolving power of 240,000, requiring an AGC target of 8e5 or a maximum injection time of 50 ms. MS1 scans included precursor ions ranging from 350–2000 m/z. The most intense ions were selected for HCD fragmentation (normalized collision energy of 25%) and subsequent MS2 analysis until the 1 second duty cycle lapsed. Dynamic exclusion was set to 10 seconds and the quadrupole isolation width was set to 0.5 Th. Only precursor ions with charge states of 2–5 were selected. MS2 spectra were analyzed in the ion trap on the “turbo” resolution setting, requiring an AGC target of 2e4 or a maximum inject time of 14 ms. MS2 scans included fragment ions ranging from 150–1350 m/z.

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Sensitive Peptide Chromatography and Orbitrap Analysis

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Each sample was resuspended in 0.2% formic acid. 1.5 μg of peptide was loaded onto a 75 um inner diameter × 360 um outer diameter column (CoAnn Technologies) packed in-house to 30 cm with 1.7 micron BEH C18 particles (Waters Corp). The peptides were chromatographically separated over a 90-min reverse phase gradient (0.2% formic acid mobile phase A, 0.2% formic acid/80% ACN mobile phase B) using a Vanquish Neo UHPLC (Thermo) at a column temperature of 50 °C and a flow rate of 320 nL/min. Eluting peptides were sprayed into an Orbitrap Eclipse using a Nanospray Flex ionization source (Thermo) at 2.2 kV. MS1 scans were recorded in the Orbitrap using a resolving power of 240,000, requiring an AGC target of 8e5 or a maximum injection time of 50 ms. MS1 scans included precursor ions ranging from 350-2000 m/z. The most intense ions were selected for HCD fragmentation (normalized collision energy of 25%) and subsequent MS2 analysis until the 1 s duty cycle lapsed. Dynamic exclusion was set to 10 s and the quadrupole isolation width was set to 0.5 Th. Only precursor ions with charge states of 2–5 were selected. MS2 spectra were analyzed in the ion trap on the “turbo” resolution setting, requiring an AGC target of 2e4 or a maximum inject time of 14 ms. MS2 scans included fragment ions ranging from 150 to 1350 m/z.

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Preparation of Packed Capillary TASF Columns

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Figure 1 shows a schematic of the instrument used for TASF separations. Capillary columns were prepared by packing 1.7 μm BEH C18 particles (Waters) into 150 μm I.D. fused-silica capillaries from Polymicro Technologies (Phoenix, AZ). Columns were fritted by sintering 2 μm, solid borosilicate spheres (Thermo Scientific, Fremont, CA) into the end of the column blank using an electrical arc. Particles were slurried in isopropanol (Spectrum) at a concentration of 50 mg/mL, sonicated for 25 minutes and packed using the downward slurry method at 27500 psi using a Model DSHF-302 pneumatic amplification pump from Haskel (Burbank, CA). The packing solvent was acetone (Sigma). Care was taken to pack columns of defined length, ca. 5-6 cm, by limiting the volume of slurry, and subsequent mass of particles, loaded into the packing system. This allowed columns to be packed leaving a 2 cm section free of stationary phase at the head of the column.

Groskreutz S.R, & Weber S.G. (2014). Temperature-assisted On-column Solute Focusing: A General Method to Reduce Pre-column Dispersion in Capillary High Performance Liquid Chromatography. Journal of chromatography. A, 1354, 65-74.

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