TMT enables relative quantitation of proteins present in multiple samples by labeling peptides with isobaric stable isotope tags that fragment upon collision-induced dissociation into reporter ions used for quantitation. In this study, TMT ten tags for ten samples (blast samples and controls) were analyzed simultaneously, avoiding run-to-run variation. Tryptic digested peptides from brain samples were labeled with TMT 10-plex reagents (Thermo Fisher) according to manufacturer’s instructions. Briefly, the TMT reagents (0.8 mg) were dissolved in 41 μL of anhydrous acetonitrile. Aliquots of samples were incubated with TMT reagents for 1 h at room temperature. The reactions were quenched by 8 μL of 5% hydroxylamine solution and reacted for 15 min. The combined TMT labelled samples were dried under SpeedVac, and then reconstituted by dilute tri-fluoroacetic acid solution followed by desalting by Oasis HLB 96-well μElution plate (Waters) prior to LC-MS/MS analysis.
Oasis hlb 96 well μelution plate
Manufactured by Waters Corporation
Sourced in United States
The Oasis HLB 96-well μElution plate is a solid-phase extraction (SPE) device designed for sample preparation in analytical workflows. The plate features a polymeric hydrophilic-lipophilic-balanced (HLB) sorbent for the extraction and purification of a wide range of analytes from complex matrices. The μElution format allows for the efficient concentration of samples in a small elution volume.
Automatically generated - may contain errors
Lab products found in correlation
Tmt 10 plex reagent, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Tqs quantiva triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Oasis hlb 96 well plate, by Waters Corporation (1 mentions)
Accuspin micro 17r, by Thermo Fisher Scientific (1 mentions)
Phos select iron affinity gel, by Merck Group (1 mentions)
Urea pellets, by Merck Group (1 mentions)
Microcon 30 kda centrifugal filter, by Merck Group (1 mentions)
Protein lobind tube, by Eppendorf (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Universal 320r, by Hettich (1 mentions)
Rotina 380 r, by Hettich (1 mentions)
Thermomixer c, by Eppendorf (1 mentions)
Iodoacetamide, by Thermo Fisher Scientific (1 mentions)
Mass spectrometry grade trypsin, by Promega (1 mentions)
Lobind microcentrifuge tubes 1.5 ml, by Eppendorf (1 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
16 protocols using oasis hlb 96 well μelution plate
1
Quantitative Proteomics Using TMT Labeling
2
Proteome Extraction and Digestion
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Cell pellets of the aerobic and anaerobic cultures were resuspended in lysis buffer composed of 100 mM triethylammonium bicarbonate containing 1% SDS and phosphatase/protease inhibitors. Yeast cells were lysed by glass bead milling by 10 cycles of 1 min shaking alternated with 1 min rest on ice. Proteins were reduced by addition of 5 mM DTT and incubation for 1 h at 37 °C. Subsequently, the proteins were alkylated for 60 min at room temperature in the dark by addition of 50 mM acrylamide. Protein precipitation was performed by addition of four volumes of ice-cold acetone (−20 °C), followed by 1 h freezing at −20 °C. The proteins were solubilized using 100 mM ammonium bicarbonate. Proteolytic digestion was performed by Trypsin (Promega), 1:100 enzyme to protein ratio, and incubated at 37 °C overnight. Solid phase extraction was performed with an Oasis HLB 96-well μElution plate (Waters) to desalt the mixture. Eluates were dried using a SpeedVac vacuum concentrator at 50 °C and frozen at −80 °C.
3
Quantifying Thymol in Mouse Serum
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A solid‐phase extraction and LC/MS method was developed and validated to quantify the concentration of thymol in mouse serum. Eugenol, a member of the phenylpropanoid class of chemical compounds, is used as an internal standard for serum samples and was extracted by a solid‐phase extraction technique using a Waters Oasis HLB 96‐well μElution plate. The extracts were then subjected to high‐performance liquid chromatography on a Phenomenex Kinetex C18 30 × 2.1 mm column and eluted by a gradient program of mobile phase A (0.1% Formic Acid in deionized water) and B (0.1% Formic Acid in methanol) with a flow rate of 0.3 mL/min. The chromatographic runtime was 7 min per injection. Compounds were determined by Thermo TQS Quantiva triple quadrupole mass spectrometer equipped with a heated electrospray ionization source in MS/MS mode.
The analytical method was validated using a Quantitative limit of 5 ng/mL and a Linearity range of 5 to 5000 ng/mL. Six levels of calibration standards were included in each calibration curve. The correlation coefficient was found to be 0.9933‐0.9998 and the method precision CV was found to be 6.8%‐9.1%. Furthermore, the method accuracy was 93%‐97% and the mean recovery for thymol was 96% and 92% for the internal standard.
The analytical method was validated using a Quantitative limit of 5 ng/mL and a Linearity range of 5 to 5000 ng/mL. Six levels of calibration standards were included in each calibration curve. The correlation coefficient was found to be 0.9933‐0.9998 and the method precision CV was found to be 6.8%‐9.1%. Furthermore, the method accuracy was 93%‐97% and the mean recovery for thymol was 96% and 92% for the internal standard.
4
Quantification of Nicotine Metabolites
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Blood was collected and immediately mixed with four volumes of methanol (with 1 μM nicotine D3 as an internal standard) to quench the enzyme. The samples were centrifuged at 10,000 rpm for 30 min, and the supernatant was transferred to clean tubes and evaporated in a rotatory evaporator Genevac. The residual was redissolved in 5% NH4OH in water and extracted by an Oasis HLB 96-well μElution Plate (Waters). The elution was evaporated in a rotatory evaporator Genevac and redissolved in Hepes buffer and 2% trifluoroacetic acid for liquid chromatography–mass spectrometry (LC-MS).
5
Comprehensive Proteomics Sample Preparation
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○ Urea pellets (Sigma, #U1250)
○ Microcon-30 kDa Centrifugal filters (Millipore, #MRCF0R030)
○ 1.5 ml and 2 ml protein-low-binding tubes
➢ e.g., Eppendorf ProteinLobind tubes (Eppendorf, #022431081 and #022431102)
○ Oasis HLB 96-well μElution plate (2 mg sorbent per well, Waters #186001828BA) for proteomic samples and Oasis HLB 96-well plate (10 mg sorbent per well, Waters #18600128) for phosphoproteomic samples
○ Empore™ SDB-RPS extraction disc (3 M, #00051115088162)
○ Empore™ C8 extraction disc (3 M, # 00051115088049)
○ PHOS-Select™ iron affinity gel (Sigma, #P9740)
○ Tools to cast SDB-RPS and C8 disks onto a pipette tip
➢ e.g. 16 gauge, Kel-F Hub NDL, 2 in, point style 3 needle (Hamilton, #90516) and plunger assembly (Hamilton, #1122-01)
○ 1.5 ml tube holder (GL Sciences, Inc. #5010-21514)
○ Thermal shaker
➢ e.g. Thermomixer C (Eppendorf, #5382000015)
○ 1.5 ml-tube centrifuges
➢ e.g. AccuSpin Micro 17R (Fisher Scientific, #13100676) for FASP
➢ e.g., Rotina 380R (Hettich, #1706-01) for StageTip spinning
○ 96-well plate centrifuge
➢ e.g. Universal 320R (Hettich, #1406-01)
○ Tube rotator
➢ e.g. SB3 (Stuart, no code provided)
○ Equipment for the determination of peptide concentration
➢ e.g., Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies, Inc.)
○ Speed vacuum lyophilisator
➢ e.g., CentriVap centrifugal vacuum concentrator (LabConco, #7810033) and CentriVap cold trap (LabConco, #7385030)
6
Isolation and Quantification of Urinary 15-F2t-Isoprostane
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[2H4]-15-F2t-IsoP (1ng/0.005 mL ethanol, internal standard) was added to a 0.050 mL aliquot of urine. The sample was diluted with 0.4 mL 5 % methanol in water containing 0.1 % formic acid (Solution A) and acidified to pH 3 with 0.1 N HCl. Solid phase extraction was performed using an Oasis HLB 96-well μElution plate (Waters, Milford, MA, USA) preconditioned with 0.4 mL methanol and 0.4 mL Solution A. Samples were loaded onto the μElution plate, and wells were washed with 0.4 mL Solution A and then 0.2 mL hexane. Analytes were eluted with 0.030 mL isopropanol/acetonitrile (1:1) into a 96-well LC/MS collection plate containing 0.030 mL LC/MS grade water in each well.
7
Proteomic Profiling with TMT Multiplexing
8
Yeast Proteome Sample Preparation
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Cell pellets were resuspended in lysis buffer composed of 100 mM
TEAB containing 1% SDS and phosphatase/protease inhibitors. Yeast
cells were lysed by glass bead milling and thus shaken 10 times for
1 min with a bead beater alternated with 1 min rest on ice. Proteins
were reduced by addition of 5 mM DTT and incubated for 1 h at 37 °C.
Subsequently, the proteins were alkylated for 60 min at room temperature
in the dark by addition of 50 mM acrylamide. Protein precipitation
was performed by addition of four volumes of ice-cold acetone (−20
°C) and proceeded for 1 h at −20 °C. The proteins
were solubilized using 100 mM ammonium bicarbonate. Proteolytic digestion
was performed by Trypsin (Promega, Madison, WI), 1:100 enzyme to protein
ratio, and incubated at 37 °C overnight. Solid phase extraction
was performed with an Oasis HLB 96-well μElution plate (Waters,
Milford, USA) to desalt the mixture. Eluates were dried using a SpeedVac
vacuum concentrator at 45 °C. Dried peptides were resuspended
in 3% ACN/0.01% TFA prior to MS-analysis to give an approximate concentration
of 250 ng per μL.
TEAB containing 1% SDS and phosphatase/protease inhibitors. Yeast
cells were lysed by glass bead milling and thus shaken 10 times for
1 min with a bead beater alternated with 1 min rest on ice. Proteins
were reduced by addition of 5 mM DTT and incubated for 1 h at 37 °C.
Subsequently, the proteins were alkylated for 60 min at room temperature
in the dark by addition of 50 mM acrylamide. Protein precipitation
was performed by addition of four volumes of ice-cold acetone (−20
°C) and proceeded for 1 h at −20 °C. The proteins
were solubilized using 100 mM ammonium bicarbonate. Proteolytic digestion
was performed by Trypsin (Promega, Madison, WI), 1:100 enzyme to protein
ratio, and incubated at 37 °C overnight. Solid phase extraction
was performed with an Oasis HLB 96-well μElution plate (Waters,
Milford, USA) to desalt the mixture. Eluates were dried using a SpeedVac
vacuum concentrator at 45 °C. Dried peptides were resuspended
in 3% ACN/0.01% TFA prior to MS-analysis to give an approximate concentration
of 250 ng per μL.
9
Isobaric Labeling and Quantification of Plasma and Brain Proteins
10
Cell Lysis and Peptide Sample Preparation
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Cells were harvested with TrypLE™ Select, washed twice with Ca/Mg-free PBS. The cell pellet was resuspended in lysis buffer (4% SDS, 0.1 M Tris, pH 7.6), boiled for 7 minutes at 95°C, sonicated (3 × 30 seconds) and centrifuged for 10 minutes at 14,000 g. Protein concentration was measured with the Pierce™ BCA Protein Assay Kit (cat# 232225, Thermo Fisher Scientific), and volume corresponding to 25 μg protein was further reduced in 0.1 M DTT and processed into peptides using filter-aided sample preparation [18 (link)]. Prior to usage, all filters were checked with a simple centrifugation step [19 (link)] in order to exclude nonretaining protein membrane filters. The peptide solutions were desalted with Oasis HLB 96-well μElution plate (cat# 186001828BA, Waters) using 0.1% formic acid (FA) and 80% acetonitrile (ACN)/0.1% FA as binding and elution buffers, respectively. Eluted peptides were vacuum dried and dissolved in 2% ACN, 1%FA prior to LC-MS analysis.
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