Proteomics using nano LC–MS/MS was performed according to a previous report (59 (link)). Proteins synthesized by iGeTT were precipitated by acetone and re-suspended in 28.5 μl of 8 M urea in 100 mM NH4HCO3. Then, the solution was reduced with 1.5 μl of 100 mM NH4HCO3 with 1 mM DTT at 25°C for 30 min, and alkylated with 10 μl of 220 mM Iodoacetamide in 100 mM NH4HCO3 at 25°C for 30 min. After the reductive alkylation, the solution was incubated with 0.5 μg of Wako Lys-C at 25°C for 3 h and diluted with 100 mM NH4HCO3 to 180 μl. Proteins were further digested with 1 μg of Trypsin Gold (Promega, Madison, WI, USA) at 37°C for overnight. After trypsin digestion, salt and organic solvent in the solution were removed by GL-Tip SDB (GL science, Tokyo, Japan) and the solution was concentrated to 20 μl by a centrifugal evaporator (TOMY SEIKO, Japan). This solution was diluted with 0.3% trifluoroacetic acid to 30 μl and put in Q-Exactive & Easy-nLC 1000 (Thermo fisher) equipped with a 12.5 cm × 75 μm C-18 separation column (Nikkyo Technos, Japan). Peptides in the solutions were analyzed by Proteome Discoverer (Thermo fisher). Below 1% of false discovery rate and observation of more than two unique peptides were criteria that we used to determine as identified proteins. All proteins listed were identified in more than two runs of nano LC–MS/MS.
Gl tip sdb
Manufactured by GL Sciences
Sourced in Japan
The GL-Tip SDB is a precision-engineered laboratory pipette tip designed for accurate and reliable liquid handling. It is constructed from high-quality materials and features a standardized design to ensure compatibility with a wide range of pipetting instruments.
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Lab products found in correlation
Trypsin lys c mix, by Promega (3 mentions)
Trypsin, by Promega (2 mentions)
Amicon ultra 10k, by Merck Group (2 mentions)
Jupiter proteo c18 column, by Phenomenex (2 mentions)
Trypsin gold, by Promega (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Centrifugal evaporator, by TOMY (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lunatic uv vis absorbance spectrometer, by Unchained Labs (1 mentions)
Mass spec grade, by Promega (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Pierce microplate bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Tripletof 6600 mass spectrometer, by AB Sciex (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Analyst software, by AB Sciex (1 mentions)
Trypsin lysc mixture, by Promega (1 mentions)
Asp n, by Promega (1 mentions)
Flexanalysis software, by Bruker (1 mentions)
Autoflex max, by Bruker (1 mentions)
21 protocols using gl tip sdb
1
Proteomic Analysis of iGeTT Proteins
2
Protein Extraction and Digestion for Mass Spectrometry
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Cell samples were prepared
as described in a previous study.17 (link) In
brief, precipitates were dissolved in 100 mM Tris-HCl (pH 8.5) containing
2% sodium dodecyl sulfate (SDS) using BIORUPTOR BR-II (SONIC BIO Co.,
Kanagawa, Japan) with settings at “High” and “30
s On/Off” cycle for a duration of 5 min. The extracted proteins
were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher
Scientific) at 1000 ng/μL. The extracts were reduced with 10
mM dithiothreitol for 30 min at 50 °C, followed by alkylation
with 30 mM iodoacetamide for 30 min at 25 °C in the dark. Protein
purification and digestion were performed using the sample preparation
(SP3) method.17 (link),18 (link) The tryptic digestion was performed
using 500 ng/μL Trypsin/Lys-C Mix (Promega, Madison, WI) overnight
at 37 °C. Cell digests were purified using GL-Tip SDB (GL Sciences,
Tokyo, Japan) according to the manufacturer’s protocol. The
peptides were dissolved again in 3% acetonitrile (ACN) containing
0.1% trifluoroacetic acid (TFA) and then quantified using a Lunatic
UV/Vis absorbance spectrometer (Unchained Labs, Pleasanton, CA).19 (link)
as described in a previous study.17 (link) In
brief, precipitates were dissolved in 100 mM Tris-HCl (pH 8.5) containing
2% sodium dodecyl sulfate (SDS) using BIORUPTOR BR-II (SONIC BIO Co.,
Kanagawa, Japan) with settings at “High” and “30
s On/Off” cycle for a duration of 5 min. The extracted proteins
were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher
Scientific) at 1000 ng/μL. The extracts were reduced with 10
mM dithiothreitol for 30 min at 50 °C, followed by alkylation
with 30 mM iodoacetamide for 30 min at 25 °C in the dark. Protein
purification and digestion were performed using the sample preparation
(SP3) method.17 (link),18 (link) The tryptic digestion was performed
using 500 ng/μL Trypsin/Lys-C Mix (Promega, Madison, WI) overnight
at 37 °C. Cell digests were purified using GL-Tip SDB (GL Sciences,
Tokyo, Japan) according to the manufacturer’s protocol. The
peptides were dissolved again in 3% acetonitrile (ACN) containing
0.1% trifluoroacetic acid (TFA) and then quantified using a Lunatic
UV/Vis absorbance spectrometer (Unchained Labs, Pleasanton, CA).19 (link)
3
Phosphopeptide Enrichment and Desalting
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Phosphopeptides were enriched at room temperature using hydroxy acid-modified metal oxide chromatography (HAMMOC), with minor modifications [168 (link)]. Titansphere® Phos-TiO columns (syringe barrel type SPE cartridge, cat. No. 5010-21291, GL Sciences Inc., Tokyo, Japan) were used according to the manufacturer’s instructions. Enriched phosphopeptides were desalted using two-step washing with a combination of GL-Tip SDB and GC columns (GL Sciences Inc., Tokyo, Japan), according to the manufacturer’s instructions. Desalted phosphopeptides were dried in a vacuum evaporator (Tomy, Tokyo, Japan), resuspended in 10 µL of 0.1% (v/v) TFA, and stored at −80 °C for later LC-MS/MS analysis.
4
In-solution Trypsin Digestion Protocol
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Following the removal of the washing buffer, beads were suspended in 2 M urea, 50 mM ammonium bicarbonate, and 10 mM DTT and incubated for 30 min at 37 °C with rotation. Iodoacetamide was added at a final concentration of 5% and beads were incubated additionally for 30 min at 37 °C with rotation in the dark. 0.5 µg of Trypsin/LysC Mix (Mass Spec Grade, Promega) was then added and incubated for 3 h at 37 °C with rotation. After diluting the buffer until urea was 1 M, 0.5 µg of Trypsin/LysC Mix was added again and incubated overnight at 37 °C with rotation. The supernatant containing digested peptides was separated from beads using a magnetic stand. The supernatant was desalinated and concentrated through GL-Tip SDB (GL Sciences, Tokyo, Japan). The pellet was suspended with 0.1% TFA and then analyzed in LC-MS/MS.
5
Acrolein-induced JIP4 Proteomic Analysis
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JIP4‐myc‐DDK‐overexpressing SH‐SY5Y cells treated with 40 μM acrolein with or without 10 μM Jak3 inhibitor VI were lysed with lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X‐100, protease inhibitor cocktail and phosphatase inhibitor cocktail). Cell lysates were reacted with anti‐flag M2 magnetic beads (Sigma‐Aldrich) for 2 h at 4°C, and then washed with lysis buffer five times. Then, JIP4 protein was eluted with 1 mg/ml flag peptide/PBS.
The sample reduction was performed for 15 min at 56°C with 10 mM DTT. Subsequent alkylation was performed in the dark for 30 min at room temperature with 55 mM iodoacetamide. After chloroform–methanol precipitation of protein, the precipitates were resuspended in 8 M urea and 0.1 M Tris–HCl, pH 8.5. Samples were diluted in 4 M urea with 0.1 M Tris–HCl, pH 8.5 and digested for 2 h with Trypsin/Lys‐C Mix (Promega), followed by dilution to 1 M urea with 0.1 M Tris–HCl, pH 8.5, overnight at 37°C. After stopping the digestion with 1% formic acid, the peptide mixture was subjected to solid‐phase extraction (GL‐Tip SDB, GL Science) for desalting, and peptide effluents were subsequently lyophilised. Lyophilised peptides were dissolved in 0.1% formic acid containing 2% acetonitrile.
The sample reduction was performed for 15 min at 56°C with 10 mM DTT. Subsequent alkylation was performed in the dark for 30 min at room temperature with 55 mM iodoacetamide. After chloroform–methanol precipitation of protein, the precipitates were resuspended in 8 M urea and 0.1 M Tris–HCl, pH 8.5. Samples were diluted in 4 M urea with 0.1 M Tris–HCl, pH 8.5 and digested for 2 h with Trypsin/Lys‐C Mix (Promega), followed by dilution to 1 M urea with 0.1 M Tris–HCl, pH 8.5, overnight at 37°C. After stopping the digestion with 1% formic acid, the peptide mixture was subjected to solid‐phase extraction (GL‐Tip SDB, GL Science) for desalting, and peptide effluents were subsequently lyophilised. Lyophilised peptides were dissolved in 0.1% formic acid containing 2% acetonitrile.
6
Protein Extraction and Digestion Protocol
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Protein was extracted using a phase transfer surfactant method as described previously.35 Briefly, cells were lysed with 50 mmol/L ammonium bicarbonate containing 12 mmol/L sodium deoxycholate, 12 mmol/L sodium N‐lauroylsarcosinate, and EDTA‐free protease inhibitor cocktail (Roche Diagnostics). Lysates were heated at 95°C for 5 minutes, sonicated, and then centrifuged at 19 000 g for 15 minutes at room temperature. Supernatants were collected and protein concentration was measured by a reducing agent compatible version of the Pierce microplate BCA protein assay kit (Thermo Fischer Scientific). Fifty micrograms of protein lysate was reduced with 10 mmol/L DTT, alkylated with 20 mmol/L iodoacetamide, and then diluted with 50 mmol/L ammonium bicarbonate, followed by digestion with 1:50 (w/w) trypsin for 18 hours at 37°C. After digestion, an equal volume of ethyl acetate containing 1% TFA was added. The mixture was vortexed for 1 minute and centrifuged at 15 600 g for 3 minutes. The upper phase was discarded. The lower aqueous phase was concentrated under vacuum and then desalted by GL‐Tip SDB according to the instructions of the manufacturer (GL Science).
7
Protease Immobilization and Tryptic Digestion
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The protease-immobilized Ni-Sepharose beads were suspended in 20 μl of 8 M urea containing 250 mM Tris-HCl (pH8.5), reduced with 25 mM tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 15 min and alkylated using 25 mM iodoacetamide at 37 °C for 30 min in the dark, both with shaking at 1200 rpm following the standard protocol. After dilution to a urea concentration of 2 M with 50 mM Tris-HCl (pH 8.5) followed by addition of 1 mM CaCl2, proteins on beads were directly digested with 0.1 μg trypsin (Promega) with shaking at 1200 rpm at 37 °C overnight. The digestion was stopped by adding 1/20 volume of 20% TFA, and then digested peptides were desalted using a GL-Tip SDB (GL Science) according to the manufacturer’s instructions. After vacuum concentration, the samples were dissolved in 20 µL of 2% acetonitrile (containing 0.1% TFA) and 7.25-µL aliquots were analyzed by nano-LC–MS/MS.
8
Peptide Extraction from Muscle Tissue
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Free peptides were extracted individually from the frozen muscle tissues (n = 3, for each group) using a tissue homogenizer Precellys 24 (Bertin Technologies, Ampère Montigny-le-Bretonneux, France). A small portion (~100 mg) of frozen tissue was placed in a 1.5 mL screw cap micro tube (Sarstedt K.K., Tokyo, Japan) containing 5 ceramic beads (f2 mm) and 1 mL of 0.25% acetic acid. Each sample was homogenized at four times for 30 s at 6000 rpm. All extracts were clarified by centrifugation at 16,000 ×g and 4 °C for 60 min. Each supernatant was passed through a 5000 MWCO filter (Vivaspin 20, Sarstedt K.K., Tokyo, Japan). All filtrates were freeze-dried, dissolved in 0.1% formic acid and desalted by GL-Tip SDB (GL Sciences, Tokyo, Japan). Desalted samples were extracted by 80% acetonitrile in 0.1% trifluoroacetic acid solution and concentrated in a vacuum evaporator VC-15 sp (TAITEC, Tokyo, Japan).
9
Affinity Purification and Mass Spectrometry of StarD7 Protein
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HEPA-1 cells were transfected with a plasmid encoding human StarD7 fused with a 3 × Flag tag at the C-terminus. After lysing the cells, StarD7 was affinity purified using anti-Flag antibody conjugated to beads (Sigma-Aldrich). Proteins were eluted from the beads with 3 × Flag peptide (Sigma-Aldrich) and separated by SDS-PAGE. Protein bands corresponding to the mature form of StarD7 (37 kDa) were excised, reduced with 10 mM dithiothreitol, alkylated with 50 mM iodoacetamide, and digested with 10 ng/μl trypsin overnight at 37 °C. Peptides were extracted from the gel by incubation with 50% acetonitrile (v/v) containing 1% formic acid, then the extracts were dried in a SpeedVac. For desalting, samples were dissolved in 0.1% formic acid, applied to a GL-Tip SDB (GL Sciences, Tokyo, Japan), and eluted with 80% acetonitrile (v/v) containing 0.1% formic acid. For LC/MS/MS experiments, samples were analyzed using a TripleTOF 6600 mass spectrometer (SCIEX, Framingham, MA) coupled to a nanoLC Eksigent 400 system comprising a reverse-phase LC with a nano column (75 μm × 15 cm ChromXP C18-CL, 3 μm, 120 Å). The MS and MS/MS spectral data were collected using Analyst software (SCIEX), and peptides were identified using protein plot software (SCIEX).
10
Glycoprotein Enrichment and Tryptic Digestion
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Glycoproteins underwent a precipitation process initiated by the addition of a triple volume of cold acetone, followed by a 16-h incubation period. Centrifugation ensued at 12,000 ×g for a 10-minute duration at 4°C. The resulting precipitate was then subject to reduction with 10 mM dithiothreitol at 56°C for 30 min and subsequent alkylation using 20 mM iodoacetamide at room temperature (25°C) in dark conditions for 40 minutes. N-glycoproteins were broken down with 1.8 µg of a trypsin/Lys-C mixture (Promega, Wisconsin, USA) for 16-h at a steady 37°C in a continuously agitating thermomixer set at 800 rpm. The glycopeptides precipitated through the addition of a quintuple volume of cold acetone, followed by a 16-h incubation period and centrifugation at 12,000 ×g for 10 minutes (25 (link)). The precipitate that formed was desalted using a GL-Tip SDB (GL Science, Tokyo, Japan) and subsequently dried utilizing a SpeedVac concentrator.
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