The non-stimulated (TgNS) and LPS stimulated (TgLPS) protein samples were separately digested by trypsinization using the protocol of Ru et al. [13 (link)]. Samples were reduced and alkylated before digestion. Briefly, protein samples were solubilized in 500μL of denaturing buffer [6M guanidine-HCl (Sigma-Aldrich)/25mM ammonium bicarbonate (Fisher Scientific), pH 8.5] and incubated with 250 μL of 1 mg/ml DTT (Sigma-Aldrich)/25 mM ammonium bicarbonate (Fisher Scientific) for 30 min at 55°C. Then, 500 μL of 1mg/mL iodoacetamide (Sigma-Aldrich)/25 mM ammonium bicarbonate (Fisher Scientific) was added, and the mixture was covered with aluminium foil and incubated for 15 min at 55°C. The reduced and alkylated protein sample went through buffer exchange with 25 mM ammonium bicarbonate (Fisher Scientific) using spin-columns with a molecular cut-off of 3 kDa (Merck Amicon Ultra 4) for 3 times. Then, 250 μL of digestion buffer (25 mM ammonium bicarbonate (Fisher Scientific)) and trypsin (Sigma-Aldrich) (ratio 1:50) were added to the sample and incubated at 37°C for 18 h. The samples were kept at -20°C or immediately analyzed.
Ammonium bicarbonate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, France, Belgium
Ammonium bicarbonate is a chemical compound with the formula (NH4)HCO3. It is a white, crystalline solid that is commonly used as a leavening agent in baking and as a source of ammonium ions in analytical and biochemical applications. Ammonium bicarbonate decomposes to release carbon dioxide and ammonia when heated, making it useful in certain chemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (32 mentions)
Acetonitrile, by Thermo Fisher Scientific (29 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (17 mentions)
Formic acid, by Merck Group (16 mentions)
Iodoacetamide, by Merck Group (13 mentions)
Iodoacetamide, by Thermo Fisher Scientific (13 mentions)
Acetic acid, by Thermo Fisher Scientific (11 mentions)
Methanol, by Thermo Fisher Scientific (10 mentions)
Dithiothreitol (dtt), by Promega (8 mentions)
Urea, by Thermo Fisher Scientific (8 mentions)
Chloroform, by Thermo Fisher Scientific (7 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (7 mentions)
Sequencing grade modified trypsin, by Promega (7 mentions)
Dithiothreitol (dtt), by Merck Group (6 mentions)
Trypsin, by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Sodium deoxycholate, by Thermo Fisher Scientific (5 mentions)
Milli q water purification system, by Merck Group (5 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (5 mentions)
Trifluoroacetic acid, by Merck Group (5 mentions)
88 protocols using ammonium bicarbonate
1
Trypsin-Assisted Protein Digestion Protocol
2
Proteomic Analysis of Extracellular Vesicles
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EVs isolated from bile were lysed in 40 µL of 2% RapiGest SF (Waters, MA, USA) in 50 mM ammonium bicarbonate (Honeywell Fluka, Thermo Fisher Scientific), supplemented with 50 mM DTT (FUJIFILM Wako Pure Chemical Corporation) and incubated at 60 °C for 30 min. The samples were then cooled to room temperature; 4 µL of 150 mM 2-iodoacetamide (Nacalai Tesque, Kyoto, Japan) was added to each sample after which the samples were incubated at room temperature for 30 min in the dark. The lysates were incubated with 1 µg/mL of Mass Spec Grade Trypsin/Lys-C Mix (Promega, WI, USA) at 37 °C overnight. Next a 4 µL of 10% TFA was added to the digested mixture, which was incubated at 37 °C for 30 min. After centrifugation at 13,000×g for 10 min at room temperature, the supernatant was lyophilized in a miVac system (Genevac Ltd, Ipswich, UK) and desalted using Pierce C-18 Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resultant peptide was eluted in 70% acetonitrile and was then lyophilized using a miVac system and stored at − 80 °C.
3
Pharmacological Inhibition of MAPK Pathways
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William’s E. basal medium (WEM) (cat. 112-033-101) was purchased from Quality Biological (Gaithersburg, MD, USA). L-glutamine (cat. TMS-002-C) and N-acetyl-L-cysteine (NAC) (cat. A9165) were purchased from Millipore Sigma (Burlington, MA, USA). Penicillin and streptomycin solution (cat. 25-512) was purchased from Genesee Scientific (San Diego, CA, USA). Dimethyl-sulfoxide (DMSO) (cat. 36480-AP), protease and phosphatase inhibitor cocktail (cat. 78440), dithiothreitol (cat. D1532), iodoacetamide (cat. A39271), ammonium bicarbonate (cat. 393210050), and trypsin (cat. 90057) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). MCLR (cat. 10007188, ≥95% purity) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (cat. 142015) was purchased from BeanTown Chemical (Hudson, NH, USA). SP600125 (SP) (JNK inhibitor, cat. HY-12275), MK2206 di-hydrochloride (MK) (AKT inhibitor, cat. HY-10358), and adezmapimod hydrochloride, SB203580 (SB) (p38 inhibitor, cat. HY-10256A) were purchased from Med Chem Express (Monmouth Junction, NJ, USA).
4
Palivizumab Protein Quantification Protocol
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For both samples and the standard mixture, the EtOH-precipitated pellets, which contained the intact palivizumab, were mixed with 100 μL of 50 mM ammonium bicarbonate (Thermo Scientific, Waltham, MA, USA), followed by the addition of 2 μL of 550 mM dithiothreitol (4 μL for the standard) (Promega, Madison, WI, USA) and incubated at 50 °C for 50 min to reduce disulfide bonds. After incubation, 4 μL of 450 mM iodoacetamide (8 μL for the standard) (Sigma Aldrich, St. Louis, MO, USA) was added and incubated at room temperature (RT, 20–23 °C) for 1 h in the dark to alkylate the thiol groups. To digest the denatured proteins into peptides, 2 μL of 1 μg/μL trypsin (4 μL for the standard) (Promega, Madison, WI, USA) was added and mixtures were incubated at 37 °C with shaking at 300 rpm overnight.
A stable isotope-labeled synthetic (SIS) peptide with 13C6 and 15N2 at the lysine (K) of LLIYDTSK (New England Peptide, Gardner, MA, USA) was spiked into each sample (3 μL of 10 ng/μL SIS peptide) and standard (10 μL of 100 ng/μL SIS peptide) and these tryptic peptides were combined with the supernatant from the EtOH precipitation step for each sample.
A stable isotope-labeled synthetic (SIS) peptide with 13C6 and 15N2 at the lysine (K) of LLIYDTSK (New England Peptide, Gardner, MA, USA) was spiked into each sample (3 μL of 10 ng/μL SIS peptide) and standard (10 μL of 100 ng/μL SIS peptide) and these tryptic peptides were combined with the supernatant from the EtOH precipitation step for each sample.
5
Proteomic Analysis of DHA and PGE2 Effects
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Docosahexaenoic
acid (DHA, 100 mg in 0.4 mL of ethanol) and prostaglandin E2 (PGE2)
ELISA kit (#514010) were purchased from Cayman Chemical (Ann Arbor,
MI). Dithiothreitol (DTT), iodoacetamide (IAA), fetal bovine albumin
(FBS), formic acid (FA, mass spectrometry grade), and monoclonal anti-β-actin
peroxidase (#A3854) were purchased from Sigma-Aldrich (St. Louis,
MO). The WST-1 assay kit was obtained from Clontech (Mountain View,
CA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin
were obtained from Life Technologies (Grand Island, NY). Antibodies
of phospho-NF-κB p65 (Rabbit mAb #3033) and NF-κB p65
(Rabbit mAb #8242) were from Cell Signaling (Beverly, MA). Anti-inducible
nitric oxide synthase (iNOS) (#ab15323) was from Abcam (Cambridge,
MA). Urea (electrophoresis grade), thiourea (electrophoresis grade),
sodium dodecyl sulfate (SDS), ammonium bicarbonate, BCA protein assay
kit, EZQ protein assay kit, SuperSignal west pico plus chemiluminescent
substrate, Restore PLUS Western blot stripping buffer, tumor necrosis
factor α (TNF-α) mouse uncoated ELISA kit (#88-7324-88),
C18 tips, dimethyl sulfoxide (DMSO), water (H2O, HPLC grade), and acetonitrile (ACN, HPLC grade) were obtained
from Thermo Fisher (Waltham, MA). Modified porcine trypsin was acquired
from Promega (Madison, WI).
acid (DHA, 100 mg in 0.4 mL of ethanol) and prostaglandin E2 (PGE2)
ELISA kit (#514010) were purchased from Cayman Chemical (Ann Arbor,
MI). Dithiothreitol (DTT), iodoacetamide (IAA), fetal bovine albumin
(FBS), formic acid (FA, mass spectrometry grade), and monoclonal anti-β-actin
peroxidase (#A3854) were purchased from Sigma-Aldrich (St. Louis,
MO). The WST-1 assay kit was obtained from Clontech (Mountain View,
CA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin
were obtained from Life Technologies (Grand Island, NY). Antibodies
of phospho-NF-κB p65 (Rabbit mAb #3033) and NF-κB p65
(Rabbit mAb #8242) were from Cell Signaling (Beverly, MA). Anti-inducible
nitric oxide synthase (iNOS) (#ab15323) was from Abcam (Cambridge,
MA). Urea (electrophoresis grade), thiourea (electrophoresis grade),
sodium dodecyl sulfate (SDS), ammonium bicarbonate, BCA protein assay
kit, EZQ protein assay kit, SuperSignal west pico plus chemiluminescent
substrate, Restore PLUS Western blot stripping buffer, tumor necrosis
factor α (TNF-α) mouse uncoated ELISA kit (#88-7324-88),
C18 tips, dimethyl sulfoxide (DMSO), water (H2O, HPLC grade), and acetonitrile (ACN, HPLC grade) were obtained
from Thermo Fisher (Waltham, MA). Modified porcine trypsin was acquired
from Promega (Madison, WI).
6
Membrane Protein Extraction and Quantification
7
Membrane protein extraction and digestion
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Optima grade or high-performance liquid chromatography grade methanol, acetonitrile and water were from Thermo Fisher Scientific (Waltham, MA) or Acros Organics (Pittsburgh, PA). Isoflurane was from Piramal Healthcare (Mumbai, India) through the University of Washington Medical Center Pharmacy. Synthetic heavy signature peptides were obtained from Pierce Biotechnology (Rockford, IL). The corresponding stable-isotope-labeled (SIL) peptides were from Thermo Fisher Scientific. ProteoExtract native membrane protein extraction kit was from Calbiochem (Temecula, CA). Ammonium bicarbonate and sodium deoxycholate were from Thermo Fisher Scientific and MP Biomedicals (Santa Ana, CA), respectively. BCA protein assay and in-solution trypsin digestion kits, iodoacetamide and dithiothreitol were obtained from Pierce Biotechnology (Rockford, IL).
8
Reagents and Solvents for Proteomic Analysis
9
Proteomic Analysis Using Triple-TOF Mass Spectrometer
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A Triple-TOF 5600 mass spectrometer from AB sciex (Framingham, MA, USA) and an ACQUITY UPLC system (Waters, Milford, MA, USA) were used for the proteomic analysis.
HPLC-grade acetonitrile, formic acid, trifluoroacetic acid, and ammonium bicarbonate were purchased from Thermo Fisher (San Jose, CA, USA); iodoacetamide (IAM) and dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO, USA). 8-plex iTRAQ labeling reagent was purchased from ABsciex (Framingham, MA, USA). Sequencing-grade trypsin was purchased from Promega (V5111, Madison, WI, USA).
HPLC-grade acetonitrile, formic acid, trifluoroacetic acid, and ammonium bicarbonate were purchased from Thermo Fisher (San Jose, CA, USA); iodoacetamide (IAM) and dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO, USA). 8-plex iTRAQ labeling reagent was purchased from ABsciex (Framingham, MA, USA). Sequencing-grade trypsin was purchased from Promega (V5111, Madison, WI, USA).
10
IgG Isolation via Affinity Chromatography
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IgG was isolated from the plasma samples by affinity chromatography, as described previously [28 (link)]. In brief, IgG was isolated in a high-throughput manner, using 96-well protein G monolithic plates (BIA Separations, Ajdovščina, Slovenia). Prior to use, monolithic plate was washed with ultrapure water maintained at 18.2 MΩ (Purelab Ultra, VWR, PA, USA) and equilibrated with phosphate-buffered saline (PBS). Plasma samples diluted 7-fold with PBS were applied to the protein G plate and washed with PBS to remove unbound proteins. IgG was eluted with 100 mM formic acid (Merck, Darmstadt, Germany) and immediately neutralized with 1 M ammonium bicarbonate (Thermo Fisher Scientific, MA, USA) to maintain the stability of IgG. The IgG eluates were dried overnight and stored at −20 °C until the deglycosylation procedure.
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