TG2 with associated proteins was immunoprecipitated from the total brain homogenate fractions using the Pierce crosslink magnetic IP kit (Fisher Scientific, Loughborough, United Kingdom) by protein A/G magnetic beads to which anti-TG2 antibody (IA12; University of Sheffield) [32 (link)] was crosslinked using disuccinimidyl suberate. Incubations of brain homogenates with the antibody-coated beads were performed for 22 h at 4 °C in constant rotation. TG2-associated proteins were subjected to reduction, alkylation and trypsin digestion directly on the beads after washing the beads three times with 50 mM TEAB. Beads were incubated for 15 h with 0.02 mg/mL of proteomics-grade trypsin (Promega) in 50 mM TEAB. Peptides were analyzed by RP-HPLC-ESI-MS/MS using a TripleTOF 6600+ mass spectrometer (SCIEX). Proteins were considered specifically associated with TG2 in WT and APP23 mice according to z-test analysis, using TG2−/− cohorts as background controls, as previously described [24 (link)].
Proteomics grade trypsin
Manufactured by Promega
Sourced in United States
Proteomics grade trypsin is a high-quality enzyme used for the digestion of proteins in proteomic studies. It is a serine protease that cleaves peptide bonds on the C-terminal side of lysine and arginine residues, generating peptides suitable for mass spectrometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Merck Group (10 mentions)
Iodoacetamide, by Merck Group (10 mentions)
Dithiothreitol (dtt), by Merck Group (8 mentions)
Hlb oasis spe cartridges, by Waters Corporation (7 mentions)
Ammonium bicarbonate, by Merck Group (7 mentions)
Acetonitrile, by Avantor (6 mentions)
Urea, by Merck Group (5 mentions)
Proteasemax surfactant, by Promega (3 mentions)
C18 cartridges, by Scharlab (2 mentions)
Silac heavy lysine lys 6, by Thermo Fisher Scientific (2 mentions)
Arg 6, by Thermo Fisher Scientific (2 mentions)
C18 ziptip, by Merck Group (2 mentions)
Tripletof 6600 mass spectrometer, by AB Sciex (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
Glucose 6 phosphate (g6p), by Merck Group (1 mentions)
Hplc grade acetonitrile, by Avantor (1 mentions)
22 protocols using proteomics grade trypsin
1
Immunoprecipitation and Mass Spectrometry of TG2 Interactome
2
Comprehensive Chemical Reagents Acquisition
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Chemical reagents and media components were purchased from Fisher Scientific (Pittsburgh, PA), Sigma Chemical Company (St Louis, MO), Difco (Sparks, MD), J.T. Baker (Center Valley, PA), Cardinal Health (Dublin, OH) and United States Biochemical (Cleveland, OH). Nitrocellulose was purchased from BioRad (Germany). HPLC solvents including acetonitrile and water were obtained from Burdick & Jackson (Muskegon, MI). Reagents for protein chemistry including iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate and formic acid were purchased from Sigma Aldrich (St. Louis, MO). HLB Oasis SPE cartridges were purchased from Waters (Milford, MA), and proteomics grade trypsin was from Promega (Madison WI). Trypsin-predigested β-galactosidase (a mass spectrometric quality control standard) was purchased from AB SCIEX (Foster City, CA).
3
Metabolomics and Proteomics Analytical Reagents
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Glucose‐6‐phosphate, citric acid, lactate, and ammonium acetate were obtained from Sigma‐Aldrich (St. Louis, MO, USA). For metabolomics studies, HPLC‐grade solvents acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA) and VWR (Radnor, PA, USA). Deionized water was generated in‐house for mobile phase preparation. For proteomics studies, HPLC solvents including acetonitrile and water were obtained from Burdick & Jackson (Muskegon, MI, USA). Reagents for protein chemistry including iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate, and formic acid were purchased from Sigma‐Aldrich. Proteomics grade trypsin was from Promega (Madison, WI, USA).
4
Protein Extraction and Preparation for Mass Spectrometry
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Lyophilized leaf tissue (1000 mg weight) was ground to a fine powder in liquid nitrogen using a pestle and mortar. Proteins were extracted from the powder by using the TCA–acetone–phenol protocol, as reported by Wang et al. [62 (link)], and then quantified with the Bradford assay, using bovine serum albumin (BSA) as the standard [63 (link)].
Protein samples (50 µg equivalent of BSA) were cleaned and concentrated by SDS–PAGE (12%), with the gel being Coomassie-stained [64 (link)]. The single band obtained was manually excised, destained, and digested with proteomics-grade trypsin (Promega, Madison, WI, USA) to a final concentration of 12.5 ng µL−1, in accordance with the method used by Romero-Rodríguez et al. [65 (link)].
Digested peptides were desalted using C18 cartridges (Scharlau, Barcelona, Spain), and eluted peptides were vacuum-dried and dissolved in a mixture of 70:30 (v/v) acetonitrile (ACN)/water containing 0.1% trifluoroacetic acid.
Protein samples (50 µg equivalent of BSA) were cleaned and concentrated by SDS–PAGE (12%), with the gel being Coomassie-stained [64 (link)]. The single band obtained was manually excised, destained, and digested with proteomics-grade trypsin (Promega, Madison, WI, USA) to a final concentration of 12.5 ng µL−1, in accordance with the method used by Romero-Rodríguez et al. [65 (link)].
Digested peptides were desalted using C18 cartridges (Scharlau, Barcelona, Spain), and eluted peptides were vacuum-dried and dissolved in a mixture of 70:30 (v/v) acetonitrile (ACN)/water containing 0.1% trifluoroacetic acid.
5
HPLC-MS Proteomics Sample Preparation
6
Bovine Protein Sample Preparation
7
Purification and Analysis of Proteins
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3× FLAG peptide was from Sigma (catalog no. F4799). HPLC solvents, including acetonitrile and water, were obtained from Burdick & Jackson (Muskegon, MI). Reagents for protein chemistry, including iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate, formic acid, and urea, were purchased from Sigma. Proteomics grade trypsin was from Promega (Madison WI). HLB Oasis SPE cartridges were purchased from Waters (Milford, MA).
8
On-Tissue Tryptic Digestion for MALDI MSI
9
Protein Sample Preparation for MS
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The protein sample was prepared in 100 μl of buffer NH4HCO3 (50 mM, pH 8.0) with 0.02% ProteaseMAX surfactant (Promega, Madison, WI, USA). After that, the sample was reduced with dithiothreitol (DTT) at 10 mM and 56°C for 20 min and alkylated with iodoacetamide 50 mM at room temperature in the dark for 20 min. One microgram of proteomics-grade trypsin (Promega) was added, and the sample was incubated at 37°C for 4 h. Finally, the sample was centrifuged at 15,000 × g for 1 min to collect the condensate, and 0.5% TFA was added to stop the digestion. Peptides were cleaned up with C18 ZipTips (Millipore) and evaporated using an Eppendorf vacuum concentrator, model 5301.
10
Russell's Viper Venom Proteomics
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Pre-cast NuPAGENovex® Bis-Tris Mini Gels, buffers and Mark 12 unstained molecular mass standards were obtained from Life Technologies, Invitrogen Inc, USA. RV (D. r. russelii) venom of Pakistan origin was a gift from the Kentucky Reptile Zoo, USA. Protein concentration standard reagents were purchased from BioRad Inc, USA. The deglycosylation mix kit was purchased from New England Biolabs, Inc, USA. The proteomics grade trypsin was obtained from Promega, USA. Lyophilized monovalent antivenom (against D. r. russelii venom) was obtained from Vins Bioproducts Limited, India (batch no: 30AS11001; expiry date: 04/2015). Cell culture media was supplied by Invitrogen Inc, USA. All other chemicals used were of analytical grade and were procured from Sigma-Aldrich, USA.
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