The materials used included hexamethyldisiloxane (HMDS, Sigma-Aldrich, St. Louis, MO, USA); sodium fluoride (NaF, Sigma Chemical, St. Louis, MO, USA); trypsin (Trypsin Gold Mass Spectrometry, Promega, Madison, USA); PlusOne 2D Cleanup kit (GE Healthcare, Uppsala, Sweden) and 3 kDa AMICON (Millipore, St Charles, MO, USA). Antibodies: nNOS (H-299, sc-8309 Santa Cruz, Dallas, TX, USA), Mouse anti-HuC/D (A-21271, Invitrogen, Waltham, MA, USA), Rabbit anti-CGRP (AB15360, Millipore, St Charles, MO, USA), Rabbit anti-VIP (V0390, Sigma-Aldrich, St. Louis, MO, USA), Goat anti-substance P (sc9758, Santa Cruz, Dallas, TX, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), Anti-mouse 488 (A21202, Invitrogen, Waltham, MA, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), and Anti-goat 568 (A11057, Invitrogen, Waltham, MAs, USA).
Trypsin gold mass spectrometry
Manufactured by Promega
Sourced in United States
Trypsin Gold Mass Spectrometry is a proteolytic enzyme used in mass spectrometry applications to cleave proteins into smaller peptides for analysis.
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Lab products found in correlation
6 protocols using trypsin gold mass spectrometry
1
Immunofluorescence Analysis of Neuronal Markers
2
Proteome Profiling of Dietary Interventions
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Protein spots from the experimental groups (control vs. WDG) were selected based on their MW and pI obtained by image analysis, cut out (fragments of approximately 1 mm3), and prepared according to the method described in [28 (link)]. In brief, the sediments were transferred to microtubes and submitted to the following four steps: (1) removal of the dye; (2) reduction and alkylation; (3) trypsin digestion (Trypsin Gold Mass Spectrometry, Promega, Madison, WI, USA); and (4) elution of peptides extracted from the gel. Subsequently, mass spectra of the peptides were obtained by analyzing the aliquots of the solutions using a nanoACQUITY UPLC-Xevo TQ-MS System (Waters, Manchester, UK). The proteins of the Bos taurus genome were identified in the UniProt database (UniProtKB/Swiss-Prot, available online: www.uniprot.org , accessed on 1 August 2022).
3
Proteomic Analysis of Salivary Glands
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The frozen salivary gland samples were homogenized. Then, protein extraction was performed by incubation in lysis buffer - 7M urea, 2M thiourea, 40 mM dithiothreitol (DTT), all diluted in an ammonium bicarbonate solution 50 mM (AMBIC) for 60 minutes under refrigeration and with continuous stirring. The samples were then centrifuged at 14,000 rpm for 30 minutes at 4° C for collection of the supernatant and thereafter, the protein content was measured in the samples pooled by the Bradford assay [21 (link)].
For 50 μL of each sample containing 50 μg of protein, 25 μL of 0.2% RapiGEST™ (Waters Co., Manchester, UK) was added and incubated at 37° C for 30 minutes. Then, 5 mM DTT was added to the solution and incubated at 37° C for 40 minutes. Subsequently, 10 mM iodoacetamide (IAA) was added and incubated for 30 minutes at room temperature under darkness. Protein digestion was performed with the addition of 10 μL trypsin (100 ng, Trypsin Gold Mass Spectrometry, Promega, Madison, USA) at 37° C overnight. Then, the samples were centrifuged at 14,000 rpm for 30 minutes at 6°C, the supernatants were collected and purified using C18 Spin columns (Pierce™). After purification, the samples were concentrated to an approximate concentration of 1 μg/μL and then, they were resuspended in 12 μL of ADH (1 pmol/μL) + 108μL of 3% acetonitrile and 0,1% formic acid for mass spectrometry analyses.
For 50 μL of each sample containing 50 μg of protein, 25 μL of 0.2% RapiGEST™ (Waters Co., Manchester, UK) was added and incubated at 37° C for 30 minutes. Then, 5 mM DTT was added to the solution and incubated at 37° C for 40 minutes. Subsequently, 10 mM iodoacetamide (IAA) was added and incubated for 30 minutes at room temperature under darkness. Protein digestion was performed with the addition of 10 μL trypsin (100 ng, Trypsin Gold Mass Spectrometry, Promega, Madison, USA) at 37° C overnight. Then, the samples were centrifuged at 14,000 rpm for 30 minutes at 6°C, the supernatants were collected and purified using C18 Spin columns (Pierce™). After purification, the samples were concentrated to an approximate concentration of 1 μg/μL and then, they were resuspended in 12 μL of ADH (1 pmol/μL) + 108μL of 3% acetonitrile and 0,1% formic acid for mass spectrometry analyses.
4
Proteomic Analysis of Th0 and Th9 Cells
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30 µg proteins from each of Th0 and Th9 cells were resolved by 10% SDS-PAGE followed by commassie blue staining. 21 gel pieces were excised for each of the Th0 and Th9 conditions and in-gel digestion was carried out as described previously19 (link),20 (link). The excised bands were destained with 40 mM ammonium bicarbonate (ABC) in 40% acetonitrile (ACN). The gel bands were processed further for reduction using 5.0 mM dithiothreitol (DTT) (60 °C for 45 min) and alkylation using 20 mM iodoacetamide (IAA) (room temperature for 10 min in dark)19 (link),20 (link). The gel sections were dehydrated with 100% ACN followed by trypsin digestion for 10–12 h at 37 °C (Gold mass-spectrometry trypsin; Promega)19 (link),20 (link). The peptides were extracted from the gel pieces with 0.4% formic acid twice, once in 50% ACN solution and once in 100% ACN. The peptides were then vacuum-dried and stored at − 80 °C for LC–MS/MS analysis19 (link),20 (link).
5
Identifying VapBC12 Toxin-Antitoxin Interactors
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BCG strain overexpressing the toxin-antitoxin (vapBC12) locus where antitoxin is His tagged was used for sample preparation. A log-phase culture of the overexpressed strain was washed with PBS and inoculated into minimal medium with 0.1% glycerol and minimal medium with 0.01% cholesterol. The cultures were allowed to grow for 48 h, and cell lysate was prepared. Immunoprecipitation was performed with an anti-His antibody (BTL1010).
In-solution digestion was carried out using 10 μg of the proteins from each condition. The samples were subjected to reduction and alkylation using 5 mM dithiothreitol (60°C for 45 min) and alkylation using 10 mM iodoacetamide. Trypsin (Gold Mass-Spectrometry Trypsin; Promega, Madison, WI) digestion was carried out at 37°C for 10 to 12 h. The peptides were vacuum-dried and stored at −80°C until LC-MS/MS analysis.
In-solution digestion was carried out using 10 μg of the proteins from each condition. The samples were subjected to reduction and alkylation using 5 mM dithiothreitol (60°C for 45 min) and alkylation using 10 mM iodoacetamide. Trypsin (Gold Mass-Spectrometry Trypsin; Promega, Madison, WI) digestion was carried out at 37°C for 10 to 12 h. The peptides were vacuum-dried and stored at −80°C until LC-MS/MS analysis.
6
SDS-PAGE Protein Digestion Protocol
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Ten micrograms of crude venom were resolved by SDS-PAGE. The gel was stained with Coomassie brilliant blue and destained with water. The specific band of ~55kDa was cut from the gel. Forty mM ammonium bicarbonate (ABC) in 40% acetonitrile (ACN) was used to destain the excised bands. Reduction and alkylation were done by 5 mM dithiothreitol (DTT) at 60 °C for 45 min and 10 mM iodoacetamide (IAA) on the gel bands respectively. The gel pieces were dehydrated using 100% ACN and dried further for 10 mins at room temperature (RT). The ingel digestion was carried out as explained previously (Goel et al., 2013) . Trypsin (Gold massspectrometry trypsin; Promega, Madison, WI) was added in ice-cold tubes and kept at 37 °C for 10-12 h. Peptides were removed from the gel pieces by adding 50% ACN with 0.1% formic acid J o u r n a l P r e -p r o o f (FA) in the tubes. Finally, the same step was carried with 100% ACN with 0.1% FA. The peptides were lyophilized and kept at -80 °C until LC-MS/MS analysis.
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