Cell pellets of approximately one million cells were resuspended in 8 M Urea (Biochemica, Billingham, UK) and 50 mM ammonium bicarbonate (AMBIC, Sigma-Aldrich) buffer at pH 8. The lysates were sonicated in the Bioruptor instrument (Diagenode, B01020001, Seraing, Belgium) for 15 cycles of 30 s at maximum mA at 4 °C. Subsequently, after centrifugation at 20,000g at 4 °C for 30 min, the soluble protein fraction was collected and the protein concentration was determined by a Bradford protein assay. Proteins were reduced with a final concentration of 5 mM DTT (Sigma-Aldrich) at 37 °C for 60 min and then alkylated for 60 min in the dark at room temperature with a final concentration of 15 mM iodoacetamide (IAA, Sigma-Aldrich). The digestion was carried out with a mixture of endoproteinase Lys-C and Trypsin (Trypsin/Lys-c Mix, Promega, Madison, WI). The first step consists of endoproteinase Lys-C digestion for 4 h at 37 °C with a protein-to-enzyme ratio of 50:1 (w/w). Next, the samples were diluted eight times with 50 mM AMBIC to a urea concentration of 1 M. The second step of digestion was performed with trypsin overnight at 37 °C with a substrate-to-enzyme ratio of 50:1 (w/w). After digestion, the samples were acidified with formic acid (FA) and desalted on C18 spin columns (Harvard Apparatus, Holliston, MA).
C18 spin column
Manufactured by Harvard Apparatus
Sourced in United States
C18 spin columns are a type of chromatography column used for the purification and separation of various compounds, including peptides, proteins, and small molecules. These columns contain a silica-based stationary phase coated with octadecyl (C18) functional groups, which provide a hydrophobic surface for the selective retention and elution of analytes. C18 spin columns are designed for rapid and efficient sample preparation, allowing for the extraction and concentration of target compounds from complex matrices.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Honeywell (2 mentions)
Sequencing grade trypsin, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ambic, by Merck Group (1 mentions)
Trypsin lys c mix, by Promega (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Iodoacetamide iaa, by Merck Group (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Q exactive orbitrap hybrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Urea, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Mass profiler professional, by Agilent Technologies (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Bullet blender, by Next Advance (1 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (1 mentions)
10 protocols using c18 spin column
1
Sample Protein Extraction and Digestion
2
Quantitative Proteomics of Small Extracellular Vesicles
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sEV protein samples were suspended in lysis buffer supplemented with a protease inhibitor cocktail and sonicated for 20 min. BCA protein assay kit (Pierce) was used to quantify protein concentration. Samples were prepared using Filter-Aided Sample Preparation (FASP) digestion method. The digested peptides were desalted using C18 spin columns (Harvard Apparatus), and the peptides were eluted with 80% acetonitrile in 0.1% formic acid in water. Peptides were analyzed with Q-Exactive Orbitrap hybrid mass spectrometer (Thermo) and Ultimate 3000 (U)HPLC systems (Thermo) to obtain proteomics data. Samples were prepared in duplicates and total of 4 runs were performed. Proteome Discoverer software (ver. 2.5) was used to identify and quantify proteins present in sEVs and Uniprot was used to download the homo sapiens database and predict the subcellular localization of the identified proteins. Fold change was calculated based on protein abundance. Gene Ontology terms associated with the genes encoding the identified proteins were analyzed to identify and group molecular functions of differentially regulated sEV proteins (fold change > 2, p-value < 0.05).
3
Cell Lysis and Protein Digestion Protocol
4
Proteomic Analysis of E. huxleyi Calcium Responses
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E. huxleyi CCMP371 cells cultured in two different calcium concentrations (0.1 and 10 mM) were harvested by centrifugation. Biologically duplicated samples of each calcium concentration were used for analysis (n = 2). Protein was reduced with 5 mM TCEP (Thermo, USA) for 30 min at 37°C and alkylated by blocking cysteine residues with 15 mM IAA (Sigma Aldrich, St. Louis, MO, USA) at 25°C for 1 h in the dark. The pH was adjusted to 8 using 1M Tris (Sigma Aldrich, St. Louis, MO, USA), and the urea (Sigma Aldrich, St. Louis, MO, USA) concentration was reduced to less than 2 M using 10 mM Tris. The reduced and alkylated proteins were digested using sequence graded trypsin at a 1:50 ratio of protein: trypsin (Promega, Madison, WI, USA) at 37°C overnight. The activity of trypsin was stopped by adding formic acid (FA), and the pH was reduced to 2–3 before desalting. The digested peptides were desalted using C18 spin columns (Harvard Apparatus, Holliston, MA, USA), and the peptides were eluted with 80% acetonitrile in 0.1% formic acid (Honeywell, Charlotte, NC, USA) in water. All high-performance liquid chromatography–grade solvents were obtained from J.T Baker (Phillipsburg, NJ, USA).
5
Hippocampal Proteomic Profiling
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Proteomic analyses were performed as described previously [18 (link)]. Briefly, hippocampal tissues were sonicated and digested with trypsin at 37 °C, followed by inactivation with formic acid (Honeywell, Dallas, TX, USA). C18 spin columns (Harvard Apparatus, Holliston, MA, USA) were used to desalt the peptides. Samples were analyzed by LC–MS/MS. Quantification of protein was performed and data were analyzed using Mass Profiler Professional (Agilent Technologies, Santa Clara, CA, USA). The detailed methodology is presented in the Supplementary File .
6
Proteomic Analysis of Grade I Meningiomas
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The twelve tissue samples of grade I meningiomas were pulverized in liquid nitrogen, as previously described78 . Then, protein extraction was made in a solution of 0.1% of RapiGest (w/v) in 50 mM triethylammonium bicarbonate (TEAB). Subsequently, the extracted proteins were centrifuged at 18,000 × g at 4 °C, for 15 minutes and supernatant was collected. The protein content was quantified by a fluorimetric assay using the Qubit 2.0 platform, according to the manufacturer’s instructions. Next, 180 µg of total protein from each sample was reduced with 10 mM of dithiothreitol (DTT) at 60 °C for 30 minutes. Then, all samples were cooled to room temperature and incubated in the dark with 25 mM of iodoacetamide (IAA) for 30 minutes. The samples were subsequently digested for 20 hours with high sequence grade modified trypsin at a 1:50 (Enzyme/Substrate) ratio at 37 °C. Following digestion, all reactions were acidified with 10% (v/v) trifluoroacetic acid (0.5% v/v final concentration) to stop proteolysis and precipitate RapiGest. The samples were centrifuged for 15 minutes at 18,000 × g at 20 °C to remove insoluble materials. Then, peptides were desalted with a C18 spin column, according to the manufacturer’s instructions (Harvard Apparatus).
7
Proteomic Analysis of ICH-Induced Brain Tissue
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Mice were deeply anesthetized and perfused transcardially with ice-cold PBS. The ipsilateral hemisphere containing both hematoma and perihematomal brain tissue was collected from the ICH animals. The respective brain area from sham animals served as the control. To study the proteomic changes that occur at the cellular level, single-cell suspensions were prepared by dissociating the brain tissue by passing through a 100-μm cell strainer using the plunger end of a 1-ml syringe and centrifuged at 1200 rpm for 5 min at 4 °C. The resultant pellet was sonicated and centrifuged at 14,000 rpm for 5 min at 4 °C and the supernatant was collected for LC-MS/MS analysis and using BCA protein assay kit (Pierce, Rockford, IL, USA) the protein concentration was estimated. One hundred micrograms of protein was reduced with dithiothreitol, alkylated using iodoacetamide, and digested overnight using sequencing-grade trypsin (catalogue number: PI90305, Thermo Scientific, USA). Digested peptides were cleaned up using C18 spin column (catalogue number: 74-4607, Harvard Apparatus, USA) and then lyophilized.
8
Proteomic Analysis of Intracerebral Hemorrhage
9
Kidney Protein Extraction and Digestion
10
Chromatin Extraction and SERPINE1 IP
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Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield.
An aliquot of chromatin (125 μg) was precleared with protein G agarose beads (Invitrogen). Proteins of interest were immunoprecipitated using 13 μg of antibody against SERPINE1 (Abnova, Cat. # H00005054-M01) and protein G magnetic beads. Protein complexes were washed then trypsin was used to remove the immunoprecipitate from beads and digested the protein sample. Protein digests were separated from the beads and purified using a C18 spin column (Harvard Apparatus). The peptides were vacuum dried using a speedvac.
An aliquot of chromatin (125 μg) was precleared with protein G agarose beads (Invitrogen). Proteins of interest were immunoprecipitated using 13 μg of antibody against SERPINE1 (Abnova, Cat. # H00005054-M01) and protein G magnetic beads. Protein complexes were washed then trypsin was used to remove the immunoprecipitate from beads and digested the protein sample. Protein digests were separated from the beads and purified using a C18 spin column (Harvard Apparatus). The peptides were vacuum dried using a speedvac.
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