Hepatocytes were boiled at 95 °C for 5 min, followed by three rounds of sonication for 10 s. The protein concentration of the lysate was determined using Pierce™ BCA protein assay kit (Thermo Scientific, #23225, USA). 100 μg of protein was reduced with 10 mmol/L tris(2-carboxyethyl)phosphine (TCEP) for 20 min at 65 °C and alkylated with 40 mmol/L chloroacetamide for 20 min in the dark. Proteins were digested with 1 μg trypsin at 37 °C in a shaking water bath overnight, the peptides acidified with formic acid to a pH of 3, diluted 1:1 (v:v) with ethyl acetate, centrifuged at 15,000×g for 5 min, and peptides purified with Millipore® C18 Ziptips (Sigma Aldrich, USA). Peptides were dried using a Speedvac concentrator (Eppendorf Concentrator Plus, Germany), reconstituted in 0.1% formic acid and 2% acetonitrile (ACN), then sonicated in a water bath for 10 min. Samples were centrifuged at 16,000×g for 5 min and, the supernatant was transferred into mass spectrometry vials and stored at 4 °C.
Millipore c18 ziptips
Manufactured by Merck Group
Sourced in United States
The Millipore C18 Ziptips are small, disposable pipette tips used for sample preparation in analytical chemistry. They contain a C18 stationary phase that can be used to extract and concentrate analytes from complex samples prior to analysis by techniques such as mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Phusion dna polymerase, by New England Biolabs (3 mentions)
Trypsin, by Worthington (3 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (3 mentions)
Lysozyme, by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (2 mentions)
Benzonase, by Merck Group (2 mentions)
Gibson assembly master mix, by New England Biolabs (2 mentions)
Nhs fluorescein, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lc ms grade water, by Thermo Fisher Scientific (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Optima grade formic acid, by Thermo Fisher Scientific (1 mentions)
Endoproteinase glu c, by Thermo Fisher Scientific (1 mentions)
Tof tof 5800 system, by AB Sciex (1 mentions)
Axima performance maldi tof tof, by Shimadzu (1 mentions)
Micro bio spin chromatography column, by Bio-Rad (1 mentions)
His60 ni superflow resin, by Takara Bio (1 mentions)
Dpni, by New England Biolabs (1 mentions)
10 protocols using millipore c18 ziptips
1
Proteomic Profiling of Hepatocytes
2
Quantitative Kynurenine Pathway Analysis
3
Phosphopeptide Enrichment and LC-MS/MS Analysis
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The enriched phosphopeptides were dissolved in NETN buffer (consisting of 0.5% NP-40, 100 mM NaCl, 50 mM Tris-HCl and 1 mM EDTA, pH 8.0) and incubated with pre-washed IMAC antibody beads (CST, Danvers, USA) with gentle shaking at 4 °C overnight. The beads were then washed with NETN buffer four times and with H2O twice. Then, 0.1% trifluoroacetic acid was used to elute the bound peptides from the beads and the eluted vacuum-dried fractions were combined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on the resulting peptides, desalted with Millipore C18 ZipTips (Sigma-Aldrich, Shanghai, China). All the experiments were conducted according to the manufacturer’s instructions. The results were analyzed using two-dimensional LC-MS/MS [20 (link)].
4
IDE Cleavage of FBG and Glucagon
5
Optimized MALDI-TOF Peptide Profiling
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The samples were mixed in a saturated aqueous solution containing sulfuric acid
(1:1 v/v) and synergistic acid (90% of 2,5-dihydroxybenzoic acid and 10% of
α-cyano-4-hydroxycarnamic acid) as described by da Silva et al. [26 (link)]. Briefly, a cation exchange step was
added immediately before the analysis on an AnchorChip 600/384 MTP plates. This
was co-crystallized at ambient temperature, and the samples were processed with
reagents from Sigma-Aldrich (USA). The α-cyano-4-hydroxycinnamic acid MALDI
matrix was processed with Millipore® C18 Ziptips. MALDI-TOF mass spectrometry
was performed on an Axima Performance MALDI-TOF/TOF (Shimadzu, Japan) using an
α-cyano-4-hydroxycinnamic acid as the matrix. The peptide profile was acquired
in linear mode with 75 V laser power.
(1:1 v/v) and synergistic acid (90% of 2,5-dihydroxybenzoic acid and 10% of
α-cyano-4-hydroxycarnamic acid) as described by da Silva et al. [26 (link)]. Briefly, a cation exchange step was
added immediately before the analysis on an AnchorChip 600/384 MTP plates. This
was co-crystallized at ambient temperature, and the samples were processed with
reagents from Sigma-Aldrich (USA). The α-cyano-4-hydroxycinnamic acid MALDI
matrix was processed with Millipore® C18 Ziptips. MALDI-TOF mass spectrometry
was performed on an Axima Performance MALDI-TOF/TOF (Shimadzu, Japan) using an
α-cyano-4-hydroxycinnamic acid as the matrix. The peptide profile was acquired
in linear mode with 75 V laser power.
6
Cloning and Expression of Streptomyces Proteins
7
Protein Extraction and Trypsin Digestion
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Frozen mycelia from PDB cultures were ground in liquid N2 and protein extracted as described previously [21 (link)] with some alterations. Briefly, 1 g of crushed mycelia was resuspended in 6 mL of lysis buffer (25 mM Tris-HCl, 6 M GdnHCl, 10 mM DTT pH 8.6) and sonicated. Protein extraction from PDA agar plates was performed as described previously [19 (link)]. All lysates were clarified twice by centrifugation, and resulting supernatants were brought to 15% TCA. Precipitated protein was pelleted by centrifugation after 30 min, and pellets were washed twice with ice-cold acetone. Pellets were finally resuspended in UT buffer (6 M urea, 2 M thiourea, 0.1 M Tris-HCl pH8). Protein concentration was determined using a Bradford protein assay, and all samples were adjusted to 1 mg/mL in advance of digestion. Trypsin digestion of protein samples was carried out as described previously [22 (link)], and sample clean-up was performed using Millipore C18 Ziptips® (Billerica, MA, USA), as per the manufacturer’s guidelines.
8
Mutagenesis and Protein Purification Protocol
9
Cloning and Expression in E. coli
10
Heterologous Expression of Streptomyces Proteins
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