Digest was injected from the autosampler of an Easy-nLC 1000 to the trapping column containing a Kasil frit and 2-cm 5 μm C18 resin (Phenomenex). High salt concentration was removed by washing with buffer A through the waste line. The analytical column is a pulled 5 um tip packed with 15to 20-cm 3 μm C18 resin (Phenomenex, Torrance, CA, USA). The peptides were injected directly onto the trapping column at a flow rate of 5.0 μL/min and separated on the analytical column at a flow rate of 500 nL/min. Buffer A was 5% acetonitrile, 0.1% formic acid, and buffer B was 80% acetonitrile, 0.1% formic acid. The gradient conditions were as follows: 0-5 min: 1-7%B; 5-95 min: 7-55%B; 95-115 min: 55-100%B; 115-120 min: 100%B; 120-125 min: 100-1%B; 125-145 min: 1%B. The eluate was sprayed at 3.0 kV and the ion transfer tube maintained at 275 °C. The mass spectrometer (LTQ Orbitrap Velos Pro) precursor scan was acquired with the AGC set to 10 5 and 50 ms maximum injection time. In all data acquisition strategies, MS2 scans were acquired by CID at a normalized collision energy set to 35 kV.
C18 resin
Manufactured by Phenomenex
Sourced in United States
C18 resin is a silica-based material commonly used in liquid chromatography. It consists of a silica gel matrix with covalently bonded C18 (octadecyl) functional groups. This resin is widely used for the separation, purification, and analysis of a variety of organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Zorbax extend c18 analytical column, by Agilent Technologies (1 mentions)
Endoproteinase lys c, by Roche (1 mentions)
Ltq xl, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Easy nlc 2, by Thermo Fisher Scientific (1 mentions)
6 protocols using c18 resin
1
Reversed-Phase Nano-LC-MS/MS Proteomic Workflow
2
Protein Fractionation and Digestion
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The samples were reduced using 5 mM TCEP for 20 min, and alkylated by 50 mM chloroacetamide for 30 min in the dark at room temperature. The proteins were first digested with endo-proteinase LysC (Roche, 11047825001, 1:100 w/w) at 35 °C for an hour. The mixture was then diluted to 2 M urea with tryptic digestion buffer (100 mM Tris-HCl, 1 mM CaCl2, pH 8.5) and the proteins were further digested by trypsin (Thermo, PRV5113, 1:20 w/w) for 20 hours at 35 °C. The resulting peptides from total crude lysate and soluble fractions were purified by STop-And-Go Extraction (STAGE) high-capacity tips using C18 resin (Phenomenex)50 (link).
Roughly 100 μg of peptides of each sample was fractionated by offline high pH reversed-phase chromatography using a Zorbax Extend-C18 analytical column, 5 μm, 4.6 × 50 mm (Agilent), on a 64 minute gradient (followed by a 21 minute equilibration with buffer A) with a 50 μL/min flow rate, where Buffer A contained 5 mM NH4HCO2, pH 10 and 2% acetonitrile and Buffer B contained 5 mM NH4HCO2, pH 10 and 90% acetonitrile. Ninety-six fractions were collected at 40 seconds/fraction. The resulting fractions were pooled in a non-contiguous manner, as previously described51 (link). The fractionated samples were pooled into 9 fractions.
Roughly 100 μg of peptides of each sample was fractionated by offline high pH reversed-phase chromatography using a Zorbax Extend-C18 analytical column, 5 μm, 4.6 × 50 mm (Agilent), on a 64 minute gradient (followed by a 21 minute equilibration with buffer A) with a 50 μL/min flow rate, where Buffer A contained 5 mM NH4HCO2, pH 10 and 2% acetonitrile and Buffer B contained 5 mM NH4HCO2, pH 10 and 90% acetonitrile. Ninety-six fractions were collected at 40 seconds/fraction. The resulting fractions were pooled in a non-contiguous manner, as previously described51 (link). The fractionated samples were pooled into 9 fractions.
3
Orbitrap Elite LC-MS/MS Proteomics Analysis
4
Immunoprecipitation-Mass Spectrometry Analysis
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5
Two-step Purification of Cross-linked Peptides
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Two-step purification of cross-linked peptides: Trx1 cross-linked complexes were purified as above His-tag purification. A total of 200 μg purified proteins were digested with Lys-C, and then digested peptides were diluted to 1 mL with incubation buffer (50 mM Tris, pH 8.0, 100 mM NaCl) and incubated with 40 μL Ni-NTA beads for 2 h. The beads were rinsed with wash buffer 1 for 3 times and incubation buffer for 2 times. On-beads digestion with Glu-C at 37°C for 8 h, and extract digested peptides with 50 μL incubation buffer for 3 times and desalting with stage-tip.
Strong cation exchange (SCX) enrichment: Trypsin digested peptides were loaded on 250 μm inter diameter column, which contains downstream 3 cm long reverse phase section (3 μm, C18 resin from Phenomenex), upstream 4 cm long SCX phase section (5 μm, resin from Whatman) and a frit at the end. Desalt with Buffer A (0.1% FA) and peptides were eluted from reverse phase to SCX phase with Buffer B (100% ACN, 0.1% FA). 12 fractions were collected from a series of concentration gradients of ammonium acetate (each was 5 μL: 75 mM, 100 mM, 150 mM, 275 mM, 375 mM, 500 mM, 650 mM, 800 mM, 900 mM, and 3 times of 1 M). These fractions were desalted, and 7 fractions (from 500 mM to 1 M_3) of 293T sample were analyzed by mass spectrometry.
Strong cation exchange (SCX) enrichment: Trypsin digested peptides were loaded on 250 μm inter diameter column, which contains downstream 3 cm long reverse phase section (3 μm, C18 resin from Phenomenex), upstream 4 cm long SCX phase section (5 μm, resin from Whatman) and a frit at the end. Desalt with Buffer A (0.1% FA) and peptides were eluted from reverse phase to SCX phase with Buffer B (100% ACN, 0.1% FA). 12 fractions were collected from a series of concentration gradients of ammonium acetate (each was 5 μL: 75 mM, 100 mM, 150 mM, 275 mM, 375 mM, 500 mM, 650 mM, 800 mM, 900 mM, and 3 times of 1 M). These fractions were desalted, and 7 fractions (from 500 mM to 1 M_3) of 293T sample were analyzed by mass spectrometry.
6
Proteomic Analysis of Enriched Samples
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Enriched samples were analyzed using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham MA, United States) connected to an Easy-nLCII (Thermo Fisher Scientific, Bremen, GA, United States) operating in positive ionization mode with voltage 2.3 kVe and temperature at 250°C. Peptides were separated with a 90 min gradient elution, with 5–40% solvent B (0.1% formic acid in acetonitrile) followed by a 20 min gradient with 40–95% solvent B at a flow rate of 200 nL/min. Columns were packed in house where the precolumn (ID 100 μm × OD 360) was packed with 5 cm of C18 resin (10 μm, Phenomenex Inc, Torrance, CA, United States) and analytical column (ID 75 μm × OD 360) was packed with 10 cm of ACQUA C18 resin (5 μm, Phenomenex Inc, Torrance, CA, United States). The mass spectrometer was programmed in the data-dependent acquisition mode with a resolution of 30,000 (400 m/z) followed by collision induced by dissociation (CID) of the 20 most intense ions. A dynamic exclusion time of 70 s was employed. The isolation window for the precursor ions was set to 2 Da, and the minimum number of ions to trigger events MS2 was 5000. The analyses were run in triplicate.
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