Bound proteins were eluted by tryptic digest: beads were resuspended in 200 mL of 50 mM ammonium bicarbonate, pH 8, with 2 mg of trypsin (Pierce), and incubated overnight at room temperature with end-over-end rotation. The digested supernatant was recovered, and beads were washed once with an additional 200 mL of 50 mM ammonium bicarbonate. The two eluates were pooled and dried by Speed-vac. Samples were desalted with C18 tip (Pierce C18 Spin Tips, Thermo Scientific cat. no. 84850) following the manufacturers’ protocol and dried by Speed-vac. Samples were then resuspended in 26 mL of 1% (vol/vol) formic acid, and centrifuged at 13,200 RPM for 20 minutes. 22 mL was loaded into a 96 well plate, two 10 mL injections were made for each sample.
C18 spin tips
Manufactured by Thermo Fisher Scientific
Sourced in United States
C18 spin tips are a type of solid-phase extraction (SPE) device used for the purification and concentration of analytes from liquid samples. These tips contain a reversed-phase C18 sorbent material that selectively retains nonpolar compounds, allowing for the removal of interfering matrix components. C18 spin tips are commonly used in sample preparation workflows prior to analytical techniques such as liquid chromatography-mass spectrometry (LC-MS).
Automatically generated - may contain errors
Lab products found in correlation
Trypsin lys c, by Promega (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Thermomixer, by Eppendorf (1 mentions)
Tmt mass tagging kits and reagents, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid chca, by Merck Group (1 mentions)
Maldi tof, by Bruker (1 mentions)
Trypsin lysc mixture, by Promega (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Mccoy s 5a media, by Thermo Fisher Scientific (1 mentions)
Htc116 cells, by American Type Culture Collection (1 mentions)
Formic acid, by Merck Group (1 mentions)
Nano pure system, by Thermo Fisher Scientific (1 mentions)
11 protocols using c18 spin tips
1
Tryptic Protein Elution and Purification
2
Tryptic Protein Elution and Purification
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Bound proteins were eluted by tryptic digest: beads were resuspended in 200 mL of 50 mM ammonium bicarbonate, pH 8, with 2 mg of trypsin (Pierce), and incubated overnight at room temperature with end-over-end rotation. The digested supernatant was recovered, and beads were washed once with an additional 200 mL of 50 mM ammonium bicarbonate. The two eluates were pooled and dried by Speed-vac. Samples were desalted with C18 tip (Pierce C18 Spin Tips, Thermo Scientific cat. no. 84850) following the manufacturers’ protocol and dried by Speed-vac. Samples were then resuspended in 26 mL of 1% (vol/vol) formic acid, and centrifuged at 13,200 RPM for 20 minutes. 22 mL was loaded into a 96 well plate, two 10 mL injections were made for each sample.
3
Immunoprecipitation for RIME Analysis
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Immunoprecipitation for RIME was carried out as described for ChIP-Seq. However, based on Mohammed et al, after washing the beads and prior to eluting, beads with antibody-bound proteins were washed twice with 50 mM NH4HCO3 and resuspended in 50 μl 50 mM NH4HCO3 containing 10 ng/μl trypsin/Lys-C (Promega) followed by overnight incubation at 37 °C with vigorous shaking in thermomixer (Eppendorf)37 (link). After digestion, beads were pelleted and supernatants containing peptides were transferred to new tubes. After mixing with 5 μl 20% heptafluorobutyric acid, peptides were desalted using C18 spin tips according to manufacturer protocol (Thermo Scientific). Desalted and dried peptides were reconstituted in 12 μl 0.1% formic acid. Peptides concentration was measured spectrophotometrically at 205 nm using NanoDrop One (Thermo Scientific). Peptides were analyzed as described above.
4
Quantitative Proteome Profiling by TMT-MS
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Small samples (100 μg) of crude proteins prepared from each tissue sample were condensed with 5 mM dithiothreitol (DTT) for 1 h at 65 ℃, and then alkylated with 11 mM iodoacetamide (IA) for 30 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM triethylammonium bicarbonate (TEAB) to urea concentration less than 2 M. Finally, freshly prepared trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) was added at a 1:50 w/w trypsin/protein ratio for the overnight protein digestion at 37 ℃.
The tryptic peptides resulting from trypsin digestion were vacuum concentrated and re-suspended in 0.5 M TEAB and processed for labeling using the TMT Mass Tagging Kits and Reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, USA). Briefly, one unit of TMT reagent (Catalog number: 90060) was thawed and reconstituted in anhydrous acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature, pooled, desalted using C18 spin tips (Thermo Fisher Scientific), speed-vacuum dried, and stored at -80 ℃ prior to LC-MS/MS analysis.
The tryptic peptides resulting from trypsin digestion were vacuum concentrated and re-suspended in 0.5 M TEAB and processed for labeling using the TMT Mass Tagging Kits and Reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, USA). Briefly, one unit of TMT reagent (Catalog number: 90060) was thawed and reconstituted in anhydrous acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature, pooled, desalted using C18 spin tips (Thermo Fisher Scientific), speed-vacuum dried, and stored at -80 ℃ prior to LC-MS/MS analysis.
5
Zooarchaeology by Mass Spectrometry
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We selected 12 unidentified bone samples (>1 cm in size) for zooarchaeology by mass spectrometry (ZooMS) analysis. We tested the protocol designed by Van Doorn et al. (2011 ) and used a warm (65 °C) ammonium bicarbonate buffer (50 mm ) to leach bone collagen without acid digestion. Then, trypsin digestion was carried out for 18 h at 37 °C using 0.5 μL of sequencing‐grade trypsin (Sigma). Enzymatic digestion was ended using 5 μL of 5% formic acid (FA), then the tryptic digests were purified and concentrated using C18 SpinTips (Thermo Scientific). Peptide elution was performed with 15 μL of 50% acetonitrile (ACN)/0.1% FA (v/v). Samples were dried overnight under a class 100 laminar flow hood. After re‐suspension, each sample (1 μL) was spotted on a target steel plate, and mixed with 1 μL α‐cyano‐4‐hydroxycinnamic acid (CHCA; Sigma) as matrix. The samples were then analysed in duplicate with MALDI‐ToF (Bruker) over a mass‐to‐charge range of 700–3500 m/z. Spectra were manually inspected and averaged using mMass (Strohalm et al., 2010 (link)), after setting a signal‐to‐noise ratio equal to 4. Taxonomic identification was performed comparing identified peptides with a database of peptide markers for all European, Pleistocene medium to large size mammals (Welker et al., 2016 (link)).
6
Proteomic Identification by Nano-LC ESI-MS/MS
7
Streptavidin Bead-Based Trypsin Digestion
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Streptavidin beads were washed twice with 1 mL 50 mM NH4HCO3 to exchange the buffer. Washed beads were then resuspended in 50 μl 50 mM NH4HCO3 containing 20 ng/μl trypsin/Lys-C (Promega) followed by overnight incubation at 37 °C with vigorous mixing in a thermoshaker (Eppendorf). After incubation beads were pelleted and supernatants were transferred to new tubes. Samples were acidified by adding 5 μl 20% heptafluorobutyric acid, incubated at room temperature for 5 min and clarified by 5-min centrifugation at 16000 g. Peptides from clarified samples were desalted using C18 spin tips (Thermo Scientific) according to manufacturer’s instructions. Desalted peptides were dried under vacuum and redissolved in 0.1% formic acid prior to LC–MS analysis. Peptide concentration was measured at 205 nm on Nanodrop One (Thermo Scientific).
8
Bacterial Protein Extraction and Digestion
9
Proteomic Analysis of HCT116 Cells
10
Cytosolic Protein Extraction and Trypsin Digestion
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Cell pellets were collected for each sample and the cells were Dounce‐homogenized in lysis buffer containing 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 1X protease inhibitor cocktail (Complete, Mini, EDTA‐free protease inhibitor cocktail tablets in EASYpack, Roche) in 10 mM Tris–HCl (pH 8.0). The samples were kept on ice throughout the entire procedure. Cell lysate was centrifuged at 1,000 × g at 4°C; the supernatant was saved as the cytosolic fraction, and the pellet was subjected to a single purification step via a sucrose cushion of 0.25 M and 0.88 M sucrose. The protein concentrations were determined using the Bradford protein assay (Bio‐Rad) and the samples were diluted to 2 mg/ml concentration; 50 μl of each sample was mixed with equal volume of trifluoroethanol, then 15 mM DTT was added and incubated at 55°C for 45 min. Next, the samples were alkylated with 55 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Then, the protein mixture was digested over night with mass spectrometry‐grade trypsin (Promega; at 1:50 v/w) at 37 °C. Tryptic digestion was halted by adding 2% formic acid (FA) and purified with C18 spintips (Thermo Scientific, HyperSep). The sample was stored at −80°C until LC‐MS/MS analysis.
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