The eluted proteins were reduced with dithiothreitol (DTT) at a final concentration of 10 mM for 30 min at room temperature and then alkylated with iodoacetamide (IAA) at a final concentration of 50 mM for 30 min in the dark. The resulting solutions were diluted 10 times with 100 mM NH4HCO3 (pH 8.5) and digested with mass spectrometry (MS)-grade trypsin (Promega, Madison, WI, United States) at an enzyme-to-substrate ratio of 1/75 (w/w) at 37°C overnight.
Ms grade trypsin
Manufactured by Promega
Sourced in United States, United Kingdom
MS-grade trypsin is a proteolytic enzyme used for the digestion of proteins in mass spectrometry (MS) applications. It has been purified and quality-controlled to meet the specific requirements of MS-based proteomic analyses.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Thermo Fisher Scientific (10 mentions)
Lys c, by Promega (6 mentions)
Iodoacetamide iaa, by Thermo Fisher Scientific (6 mentions)
Rapigest sf surfactant, by Waters Corporation (6 mentions)
Trifluoroacetic acid, by Merck Group (5 mentions)
Iodoacetamide, by Merck Group (5 mentions)
Dithiothreitol (dtt), by Merck Group (5 mentions)
Pierce hela protein digest standard, by Thermo Fisher Scientific (4 mentions)
Lys c ms grade, by Promega (4 mentions)
Ammonium bicarbonate, by Merck Group (3 mentions)
Rapigest, by Waters Corporation (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lys c, by Fujifilm (3 mentions)
Dithiothreitol (dtt), by Roche (3 mentions)
Formic acid, by Merck Group (3 mentions)
Trypsin, by Promega (3 mentions)
Ammonium formate, by Merck Group (2 mentions)
Iodoacetamide, by GE Healthcare (2 mentions)
N dodecyl β d maltoside ddm, by Merck Group (2 mentions)
Precast gel, by Thermo Fisher Scientific (2 mentions)
67 protocols using ms grade trypsin
1
Protein Reduction, Alkylation, and Trypsin Digestion
2
Immunoprecipitation and Immunofluorescence Protocol
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Chemicals and HPLC solvents, unless indicated otherwise in the text, were purchased from Thermo Fisher. The highest available grades were used. The following antibodies were used for Immunoprecipitation and indirect immunofluorescence: mouse monoclonal anti-GFP (B-2, Santa Cruz, CA), rabbit polyclonal anti-Numa1 (H300, Santa Cruz, CA), mouse monoclonal anti-Scribble (C6, Santa Cruz, CA), goat anti-mouse Alexa Fluor 467 (Invitrogen, USA), goat anti-rabbit Texas red-conjugated (Jackson IR laboratories). Protein A/G coated magnetic microparticles (Thermo Fisher). MS-grade trypsin was used for protein digestion (Promega).
3
Proteomics Sample Preparation
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RNA-bound proteins were treated with 100 mM NH4HCO3 containing ∼6 ng/μl of MS-grade trypsin (Promega) and incubated at 37°C overnight for 12–18 h. The samples were then vacuum dried before being submitted to MS analysis.
4
Proteomic Profiling of Triple-Negative Breast Cancer
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The dedicated procedure of sample preparation was performed (15 (link)) (Supplementary Methods , available online). Tissue samples were cut into 8-µm frozen sections and then microdissected to obtain approximately 4000 breast cancer epithelial cells. Proteins were extracted from microdissected cells, and this was followed by denaturation, reduction, and alkylation. Protein samples were digested at 37oC for 4 hours using MS-grade trypsin (Promega, Madison, WI) at a 1:4 (enzyme/protein) ratio and then acidified for further analysis.
Global proteome profiles of the TNBC samples were recorded on an nLC hyphenated LTQ-Orbitrap-XL MS system (ThermoElectron, Bremen, Germany) (Supplementary Methods , available online). Peptide mixtures were separated on the nLC system with a 3-hour binary gradient (mobile phase A: water; mobile phase B: acetonitrile) in a 3-μm C18 silica-packed 50-cm capillary column with 75-μm inner diameter. Mass spectra were acquired over a mass-to-charge ratio (m/z) range of 400 to 1800 at a resolving power of 30000 at 400 m/z. The five most intensive parent ions from the full scan were isolated and fragmented by collisional activated dissociation in the linear ion trap. Dynamic exclusion was used to increase the number of parent ions selected for fragmentation. Recorded raw nLC-MS/MS data have been submitted to ProteomeXchange (accession number: PXD000260).
Global proteome profiles of the TNBC samples were recorded on an nLC hyphenated LTQ-Orbitrap-XL MS system (ThermoElectron, Bremen, Germany) (
5
Quantitative Proteomic Analysis of Cell Signaling
6
In-Gel Digestion of Immunoisolates
7
Protein Extraction and Proteolytic Digestion
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The obtained proteins were subjected to chloroform–methanol extraction. The samples were equilibrated at room temperature for 10 min and then centrifuged at 16,000× g for 15 min at 4 °C. The pellet was washed three times in cold 80% acetone and allowed to dry in a rotary concentrator.
The samples were resuspended in 30 µL of 8 M urea and 25 mM NH4HCO3. Subsequently, the samples were reduced with 20 mM DTT and incubated for 1 h at room temperature. Then, 20 mM iodoacetamide was added, and the samples were incubated for 1 h at room temperature in the dark. After that, the samples were diluted in a 25 mM NH4HCO3 solution.
Protein digestion was performed using MS-grade trypsin (#V5071, Promega, Madison, WI, USA) at a 1:50 (protease/protein; m/m) ratio for 16 h at 37 °C. The reaction was stopped by the addition of 10% formic acid. Then, the samples were subjected to Clean Up Sep-Pak C18 Spin Columns following the manufacturer’s instructions. Subsequently, the clean peptides were dried using a rotary concentrator at 1000 rpm and 10 °C overnight.
The samples were resuspended in 30 µL of 8 M urea and 25 mM NH4HCO3. Subsequently, the samples were reduced with 20 mM DTT and incubated for 1 h at room temperature. Then, 20 mM iodoacetamide was added, and the samples were incubated for 1 h at room temperature in the dark. After that, the samples were diluted in a 25 mM NH4HCO3 solution.
Protein digestion was performed using MS-grade trypsin (#V5071, Promega, Madison, WI, USA) at a 1:50 (protease/protein; m/m) ratio for 16 h at 37 °C. The reaction was stopped by the addition of 10% formic acid. Then, the samples were subjected to Clean Up Sep-Pak C18 Spin Columns following the manufacturer’s instructions. Subsequently, the clean peptides were dried using a rotary concentrator at 1000 rpm and 10 °C overnight.
8
LC/MS Sample Preparation Protocols
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For LC/MS sample preparation, LC/MS-grade water and acetonitrile (ACN) were used, if not otherwise stated, and were purchased together with acetone from Merck, Darmstadt, Germany. Triethylammonium bicarbonate buffer (TEAB), formic acid (FA), EDTA tris(2 carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA) and trifluoroethanol (TFE) were purchased from Sigma-Aldrich, Taufkirchen, Germany. SDS was purchased from Serva Electrophoresis GmbH, Heidelberg, Germany. TFA was obtained from Roth, Karlsruhe, Germany. Rapigest was obtained from Waters, Milford, United States. 15N-labeled αSyn was purchased from rPeptide, Watkinsville, United States. MS-grade trypsin and LysN were obtained from Promega, Madison, United States. TMT10plex and TMT11-131C were purchased from Thermo Fisher Scientific, Bleiswijk, Netherlands.
9
Protein Preparation for Mass Spectrometry
10
Protein Extraction and Purification
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Ethylenediaminetetraacetic acid (EDTA), tributylphosphine (TBP), bovine serum albumin (BSA), SP, BK, NKA and NPY were purchased from Sigma (Australia). MS grade trypsin was purchased from Promega (USA). Acrylamide was purchased from Bio-Rad (USA). Immunofluorescence dyes, pre-cast gels, buffers, molecular weight markers and all standard molecular biology reagents were purchased from Life Technologies (Australia) unless otherwise noted.
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