We showcased our method using a TMT10-plex of yeast (S. cerevisiae wild-type strain BY4716) grown in synthetic complete media supplemented with 2% glucose (n=5) or 2% pyruvate (n=5) as the carbon source. We harvested the cells at OD600nm=0.8. Cells were lysed by bead-beating in 8 M urea 200mM EPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid), pH 8.5 and with protease and phosphatase inhibitors. Protein concentration was determined with the BCA assay. The BCA assay was performed according to manufacturer’s instructions with samples that were diluted at least 1:20, to ensure that the 8M urea has been diluted far below its compatibility limit. Samples were reduced with 5mM TCEP, alkylated with 10 mM iodoacetamide that was quenched with 10 mM DTT. A total of 100 μg of protein were chloroform-methanol precipitated. Protein was reconstituted in 200 mM EPPS pH 8.5 and digested by Lys-C overnight and trypsin for 6 h, both at a 1:100 protease-to-peptide ratio. Directly to the digest, we added a final volume of 30% acetonitrile and labelled 100 μg of peptide with 200 μg of TMT. To check mixing ratios, 2 μg of each sample were pooled, desalted, and analyzed by mass spectrometry. Using normalization factors calculated from this “label check,” samples were mixed 1:1 across all channels and desalted using a 100 mg Sep-Pak solid phase extraction column. The Pierce High-Select Fe-NTA Phosphopeptide Enrichment Kit was used to enrich phosphopeptides from the pooled TMT-labeled mixture. The unbound fraction and washes from this enrichment were combined and fractionated with basic pH reversed-phase (BPRP) HPLC, collected in a 96-well plate and combined down to 12 fractions prior to desalting and subsequent LC-MS/MS processing (14 (link), 15 ).
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Procedures
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Laboratory Procedure
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Solid Phase Extraction
Solid Phase Extraction
Solid Phase Extraction (SPE) is a widely used technique in analytical chemistry and biochemistry for the purification, concentration, and isolation of target analytes from complex matrices.
SPE involves the selective partitioning of analytes between a solid sorbent and a liquid phase, allowing for efficient sample preparation and cleanup prior to instrumental analysis.
This process optimizes and streamlines workflows, enhancing the reproducibility and sensitivity of downstream analyses.
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Easily locate the best protocols from literature, preprints, and patents using the AI-driven comparisons, and identify the top products and methods to enhance your SPE workflow.
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SPE involves the selective partitioning of analytes between a solid sorbent and a liquid phase, allowing for efficient sample preparation and cleanup prior to instrumental analysis.
This process optimizes and streamlines workflows, enhancing the reproducibility and sensitivity of downstream analyses.
PubCompare.ai, the leading AI platform for reproducible research, offers a seamless solution to optimize your SPE process.
Easily locate the best protocols from literature, preprints, and patents using the AI-driven comparisons, and identify the top products and methods to enhance your SPE workflow.
Experience a one-stop solution for Solid Pahse Extraction success with PubCompare.ai.
Most cited protocols related to «Solid Phase Extraction»
acetonitrile
Acids
Biological Assay
Carbon
Cells
Chloroform
ferric nitrilotriacetate
Glucose
High-Performance Liquid Chromatographies
inhibitors
Iodoacetamide
Mass Spectrometry
Methanol
Peptide Hydrolases
Peptides
Phosphopeptides
Phosphoric Monoester Hydrolases
Proteins
Pyruvate
Saccharomyces cerevisiae
Solid Phase Extraction
Staphylococcal Protein A
Strains
Tandem Mass Spectrometry
tris(2-carboxyethyl)phosphine
Trypsin
Urea
Yeast, Dried
ammonium bicarbonate
beta-glycerol phosphate
Breast
Buffers
Cells
Cholera Toxin
Cloning Vectors
Cortisone
Digestion
Enzymes
Epithelial Cells
Equus caballus
Fractionation, Chemical
Homo sapiens
Insulin
Penicillins
Peptides
Protease Inhibitors
Proteins
Serum
sodium pyrophosphate
Sodium Vanadate
Solid Phase Extraction
Streptomycin
Tablet
Trypsin
Urea
Protein content was measured using a BCA assay (Thermo Scientific); disulfide bonds were reduced with DTT and cysteine residues alkylated with iodoacetamide as previously described.10 (link) Protein lysates were cleaned up by methanol-chloroform precipitation and digested overnight with Lys-C (Wako) in a 1/100 enzyme/protein ratio in 4 M urea and 50 mM Tris-HCl, pH 8.8. The digest was acidified with formic acid (FA) to a pH of ~ 2–3 and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters).
Isobaric labeling of the peptides was accomplished with 6-plex TMT reagents (Thermo Scientific). Reagents, 0.8 mg, were dissolved in 40 μl acetonitrile (ACN), and 10 μl of the solution was added to 100 μg of peptides dissolved in 100 μl of 50 mM HEPES, pH 8.5. We found that the generation of unidentified and unwanted side reaction products – singly charged ions with m/z of 303.26, 317.26, and 331.29 – was prevented by using a 200 mM HEPES pH 8.5 buffer instead of the triethylammonium bicarbonate (TEAB) buffer recommended by the manufacturer. After 1 h at room temperature, the reaction was quenched by adding 8 μl of 5% hydroxylamine. Yeast peptides were labeled with all six reagents (126–131), human peptides were labeled with reagents 126, 127, and 128. Labeled peptides from yeast and human were separately mixed in the ratios described in the main manuscript and subjected to C18 SPE on Sep-Pak cartridges. After individual LC-MS2 analysis of both samples (Supplementary Fig. 5 ), they were mixed to generate the final multi-proteome digest mixture. Two samples were prepared using the ratios presented in Fig. 1 : one was used to study the influence of isolation specificity and precursor ion isolation width on the interference effect (see below and main manuscript), while all other experiments used the second sample. One sample was prepared using the ratios given in Supplementary Fig. 1 .
Isobaric labeling of the peptides was accomplished with 6-plex TMT reagents (Thermo Scientific). Reagents, 0.8 mg, were dissolved in 40 μl acetonitrile (ACN), and 10 μl of the solution was added to 100 μg of peptides dissolved in 100 μl of 50 mM HEPES, pH 8.5. We found that the generation of unidentified and unwanted side reaction products – singly charged ions with m/z of 303.26, 317.26, and 331.29 – was prevented by using a 200 mM HEPES pH 8.5 buffer instead of the triethylammonium bicarbonate (TEAB) buffer recommended by the manufacturer. After 1 h at room temperature, the reaction was quenched by adding 8 μl of 5% hydroxylamine. Yeast peptides were labeled with all six reagents (126–131), human peptides were labeled with reagents 126, 127, and 128. Labeled peptides from yeast and human were separately mixed in the ratios described in the main manuscript and subjected to C18 SPE on Sep-Pak cartridges. After individual LC-MS2 analysis of both samples (
acetonitrile
Biological Assay
Buffers
Chloroform
Cysteine
Disulfides
Enzymes
formic acid
HEPES
Homo sapiens
Hydroxylamine
Iodoacetamide
isolation
link protein
Methanol
M protein, multiple myeloma
Peptides
Proteins
Proteome
Solid Phase Extraction
triethylammonium bicarbonate
Tromethamine
Urea
Yeast, Dried
Samples were prepared using a modified crude extraction procedure that was originally reported by Pan et al. [27 (link)]. Three replicates of each frozen leaf sample (~100 mg for each replicate) were ground to a fine power in liquid nitrogen using a mortar and pestle. Each sample was weighed into a 1.5-mL tube, mixed with 750 μL cold extraction buffer (methanol:water:acetic acid, 80:19:1, v/v/v) supplemented with internal standards, 10 ng 2H6ABA, 10 ng DHJA, 5 ng D2-IAA, and 3 μg NAA, vigorously shaken on a shaking bed for 16 h at 4°C in dark, and then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was carefully transferred to a new 1.5-mL tube and the pellet was remixed with 400 μL extraction buffer, shaken for 4 h at 4°C, and centrifuged. The two supernatants were combined and filtered using a syringe-facilitated 13-mm diameter nylon filter with pore size 0.22 μm (Nylon 66; Jinteng Experiment Equipment Co., Ltd, Tianjing, China). The filtrate was dried by evaporation under the flow of nitrogen gas for approximately 4 h at room temperature, and then dissolved in 200 μL methanol. A aliquot of dissolved sample was further diluted 100 times using methanol for quantification of SA, because rice contains a high level of SA.
To determine whether different types of filters would influence the recovery rates of analytes, each standard (5 ng) was added into a 1.5-mL tube, mixed with the same amount of cold extraction buffer, vigorously shaken on a shaking bed, and centrifuged as described above. The samples were then filtered using a nylon filer or a syringe-facilitated 13-mm diameter cellulose filter with pore size 0.22 μm (MCE; Navigator Lab Instrument Co., Ltd, Tianjing, China). The filtrates were dried and then dissolved in methanol as described above.
For comparison, samples were also prepared using a solid-phase extraction procedure [16 (link)]. In brief, ground sample powder was mixed with 2 ml extraction buffer and shaken on a shaking bed for 16 h as for the above-described preparation for crude extraction, and then centrifuged at 3500 g for 15 min at 4°C for collecting the supernatant. The supernatant was purified using a C18-SepPak cartridge (Waters Corporation, Milford, MA, USA) by a series of steps. The purified sample was dried by evaporation and then dissolved in 200 μL of methanol as described above for preparation for the crude extraction.
To determine whether different types of filters would influence the recovery rates of analytes, each standard (5 ng) was added into a 1.5-mL tube, mixed with the same amount of cold extraction buffer, vigorously shaken on a shaking bed, and centrifuged as described above. The samples were then filtered using a nylon filer or a syringe-facilitated 13-mm diameter cellulose filter with pore size 0.22 μm (MCE; Navigator Lab Instrument Co., Ltd, Tianjing, China). The filtrates were dried and then dissolved in methanol as described above.
For comparison, samples were also prepared using a solid-phase extraction procedure [16 (link)]. In brief, ground sample powder was mixed with 2 ml extraction buffer and shaken on a shaking bed for 16 h as for the above-described preparation for crude extraction, and then centrifuged at 3500 g for 15 min at 4°C for collecting the supernatant. The supernatant was purified using a C18-SepPak cartridge (Waters Corporation, Milford, MA, USA) by a series of steps. The purified sample was dried by evaporation and then dissolved in 200 μL of methanol as described above for preparation for the crude extraction.
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Acetic Acid
Buffers
Cellulose
Cold Temperature
DNA Replication
Freezing
Methanol
Nitrogen
Nylons
Oryza sativa
Plant Leaves
Powder
Preparation H
Solid Phase Extraction
Syringes
The two-proteome interference
model was prepared as previously.14 (link),16 (link) HeLa S3 cells
were grown in suspension to 1 × 106 cells/mL. Yeast
cells were grown to an OD of 1.0. Cells were lysed in 6 M guanidiumthiocyanate,
50 mM Hepes (pH 8.5, HCl). Protein content was measured using a BCA
assay (Thermo Scientific), disulfide bonds were reduced with dithiothreitol
(DTT), and cysteine residues were alkylated with iodoacetamide as
previously described.17 (link) Protein lysates
were cleaned with methanol–chloroform precipitation.18 (link) The samples were redissolved in 6 M guanidiumthiocyanate,
50 mM Hepes pH 8.5, and diluted to 1.5 M guanidium thiocyanate, 50
mM Hepes (pH 8.5). Both lysates were digested overnight with Lys-C
(Wako) in a 1/50 enzyme/protein w/w ratio. Following digestion, the
sample was acidified with TFA to a pH < 2 and subjected to C18 solid-phase extraction (SPE, Sep-Pak, Waters).
The
TMT reagents were dissolved in 40 μL of acetonitrile, and 10
μL of the solution was added to 100 μg of peptides dissolved
in 100 μL of 50 mM HEPES (pH 8.5). After incubating for 1 h
at room temperature (22 °C), the reaction was quenched by adding
8 μL of 5% w/v hydroxylamine. Following labeling, the sample
was combined in desired ratios. Yeast aliquots were mixed at 10:4:1:1:4:10,
and HeLa was mixed at 1:1:1:0:0:0 (Figure1 A). Those two samples were then mixed at a 1/1 w/w ratio and subjected
to C18 solid-phase extraction.
model was prepared as previously.14 (link),16 (link) HeLa S3 cells
were grown in suspension to 1 × 106 cells/mL. Yeast
cells were grown to an OD of 1.0. Cells were lysed in 6 M guanidiumthiocyanate,
50 mM Hepes (pH 8.5, HCl). Protein content was measured using a BCA
assay (Thermo Scientific), disulfide bonds were reduced with dithiothreitol
(DTT), and cysteine residues were alkylated with iodoacetamide as
previously described.17 (link) Protein lysates
were cleaned with methanol–chloroform precipitation.18 (link) The samples were redissolved in 6 M guanidiumthiocyanate,
50 mM Hepes pH 8.5, and diluted to 1.5 M guanidium thiocyanate, 50
mM Hepes (pH 8.5). Both lysates were digested overnight with Lys-C
(Wako) in a 1/50 enzyme/protein w/w ratio. Following digestion, the
sample was acidified with TFA to a pH < 2 and subjected to C18 solid-phase extraction (SPE, Sep-Pak, Waters).
The
TMT reagents were dissolved in 40 μL of acetonitrile, and 10
μL of the solution was added to 100 μg of peptides dissolved
in 100 μL of 50 mM HEPES (pH 8.5). After incubating for 1 h
at room temperature (22 °C), the reaction was quenched by adding
8 μL of 5% w/v hydroxylamine. Following labeling, the sample
was combined in desired ratios. Yeast aliquots were mixed at 10:4:1:1:4:10,
and HeLa was mixed at 1:1:1:0:0:0 (Figure
to C18 solid-phase extraction.
acetonitrile
Cells
Chloroform
Cysteine
Digestion
Disulfides
Dithiothreitol
Enzymes
HeLa Cells
HEPES
Hydroxylamine
Iodoacetamide
link protein
Methanol
Peptides
Proteins
Proteome
Saccharomyces cerevisiae
Solid Phase Extraction
Staphylococcal Protein A
Thiocyanates
Most recents protocols related to «Solid Phase Extraction»
Example 1
The present example described the preparation of an HMG glucoside for use in a flavor composition through the hydrolysis of cocoa bean liquor made from West African cocoa beans.
Reagents: A solution of 4N HCl was prepared by adding 100 mL 34-37% HCl in a 250 mL volumetric flask and filling it with water. A solution of 4N NaOH was prepared by dissolving 80 g NaOH pellets in 500 mL of water in a volumetric flask.
Method: Cocoa liquor was run through a sieve and 30.09 g of fine powder was weighed into a 500 mL 3-neck round-bottom flask. The liquor was dissolved in 4N HCl (200 mL) and a stir bar was added to the flask. The sample was stirred at room temperature until the liquor was fully dispersed and flowed freely. A condenser was affixed to the flask and held at 8° C. A digital thermometer was pierced through a rubber stopper to measure the temperature of the solution. The third neck was plugged with a rubber stopper. The flask was wrapped in aluminum foil and heated to approximately 106° C. using a heating mantle. The sample was refluxed for 4.5 hours and left to cool to room temperature. The sample was transferred to a 1 L beaker and neutralized to pH 7 with 4N NaOH using a digital pH meter (pH 6.98 @29° C.). The sample was divided equally into 4 250 mL centrifuge tubes and centrifuged for 10 minutes @ 4500 rpm. The supernatant was filtered under vacuum through a Buchner funnel. The filtrate was then transferred to 2 32 oz plastic containers and lyophilized (yield 52.50 g).
1. Hydrolysis of Cocoa Powder
-
- Preparation: A solution of 4N HCl was prepared by adding 100 mL 34-37% HCl in a 250 mL volumetric flask and filling it to the line with water. A solution of 4N NaOH was prepared by dissolving 80 g NaOH pellets in 500 mL of water in a volumetric flask.
- Procedure: Cocoa liquor made from Theobroma cacao cocoa beans was run through a sieve and 30.09 g of fine powder was weighed into a 500 mL 3-neck round-bottom flask. The liquor was dissolved in 4N HCl (200 mL) and a stir bar was added to the flask. The sample was stirred at room temperature until the liquor was fully dispersed and flowed freely. A condenser was affixed to the flask and held at 8° C. A digital thermometer was pierced through a rubber stopper to measure the temperature of the solution. The third neck was plugged with a rubber stopper. The flask was wrapped in aluminum foil and heated to approximately 106° C. using a heating mantle. The sample was refluxed for 4.5 hours and left to cool to room temperature. The sample was transferred to a 1 L beaker and neutralized to pH 7 with 4N NaOH using a digital pH meter (pH 6.98 @ 29° C.). The sample was divided equally into 4 250 mL centrifuge tubes and centrifuged for 10 minutes @ 4500 rpm. The supernatant was filtered under vacuum through a Buchner funnel. The filtrate was then transferred to 2 32 oz plastic containers and lyophilized.
2. Ethanol Extraction of Hydrolyzed Cocoa Powder
-
- The hydrolyzed cocoa powder was extracted with ethanol to remove a bulk of the salts generated during neutralization. Hydrolyzed cocoa powder (50.36 g) was divided equally into 2 500 mL centrifuge tubes. Ethanol (200 mL) was added slowly to each tube as to not disturb the sample. The samples were shaken for 15 minutes on an autoshaker and then centrifuged for 10 minutes @4500 rpm. The supernatant was decanted into a 1000 mL round-bottom flask. The residue was scraped off the bottom of the tubes and redissolved in ethanol (200 mL each). The samples were shaken for 15 minutes on an autoshaker and then centrifuged for 10 minutes @ 4500 rpm. The supernatant was combined with the previous supernatant and evaporated under reduced pressure to remove all organic solvent. The remaining solids were redissolved in approximately 100 mL deionized water and lyophilized.
3. SPE (Solid Phase Extraction) Fractionation of HCP (Hydrolysed Cocoa Powder) Ethanol Extract
-
- The extract previously obtained was further fractionated to exhaustively remove the salts and hydrophilic molecules. HCP ethanol extract was transferred to 14 glass vials (approximately 0.5 g each, 20 mL volume) and dissolved in DI water (10 mL). The samples were shaken until dissolved (approximately 1 minute). The samples were filtered through a syringe and PTFE filter to remove particulates as necessary. A solid phase extraction (SPE) cartridge (20 g/60 mL, C18 stationary phase) was conditioned sequentially with DI water (100 mL), methanol (100 mL), and DI water (100 mL). The sample (10 mL) was then loaded onto cartridge and washed with DI water (100 mL) and extracted with methanol (100 mL). The cartridge was reconditioned and the remaining 13 samples were washed and extracted as previously described. The organic solutions were combined and rotary evaporated under reduced pressure. The residue was redissolved in DI water and lyophilized using a Labconco freeze dryer. The sample was separated by high-performance liquid chromatography (HPLC) to narrow down the taste-active molecules of interest.
1. Liquid/Solid Extraction of Liquor
-
- Cocoa Liquor made from cocoa beans sourced from Papua New Guinea (PNG liquor) (600 g) was frozen in liquid nitrogen and ground into a fine powder with a laboratory mill. The powder was divided equally into six plastic centrifuge tubes (500 mL volume). Each sample (100 g PNG liquor) was extracted with diethyl ether (200 mL) for 15 minutes using an autoshaker to remove the fat. After centrifugation (10 min, 4500 rpm), the supernatant was discarded. The extraction process was repeated three more times for a total of four times. The remaining defatted liquor was left to air dry in a fume hood overnight. Defatted liquor (200 g) was divided equally between four plastic centrifuge bottles (250 mL volume). To each sample (50 g defatted PNG liquor), 150 mL 70:30 acetone:water was added. The bottles were placed on an autoshaker for 15 minutes. Each sample was centrifuged (5 min, 3500 rpm) and then the supernatant was vacuum filtered using Whatman 540 filter paper and a Buchner funnel. The residue was freed from the bottom of the bottles by hand and additional 70:30 acetone:water (100 mL) was added to each sample. The samples were shaken for 15 minutes using an auto-shaker. After centrifugation (10 min, 4500 rpm), the supernatant was vacuum filtered again using the same procedure described above. The supernatants from each extraction were combined (˜800 mL) and the residue was discarded. The supernatant was rotary evaporated under reduced pressure and the remaining aqueous solution (˜250 mL) was transferred into a separatory funnel (1000 mL volume). The aqueous solution was washed with Dichloromethane (3×300 mL) to remove any xanthines. The dichloromethane layer was discarded, then the aqueous solution was washed sequentially with n-butyl acetate (3×300 mL), ethyl acetate (3×300 mL), and methyl acetate (3×300 mL) to remove procyanidins. The organic layers were discarded and the aqueous solution (F7) was rotary evaporated under reduced pressure to remove any remaining solvent. The remaining water solution was lyophilized using a Labconco freeze dryer (100×103 mbar, −40° C.). Sensory analysis was performed and the savory attribute was found to be in F7.
2. Solid Phase Extraction (SPE)
-
- For removal of any residual salts, treated PNG liquor powder (F7) was transferred to 14 glass vials (20 mL volume, approximately 0.5 g sample in each vial) and dissolved in DI water (10 mL). The samples were shaken until dissolved (approximately 1 minute). A solid phase extraction (SPE) cartridge (20 g/60 mL, C18 stationary phase) was conditioned sequentially with DI water (100 mL), methanol (100 mL), and DI water (100 mL). The vacuum was broken and the sample (10 mL) was then loaded onto cartridge. The vacuum was resumed and the sample was washed with DI water (100 mL). The receptacle flask was changed and the sample was extracted with methanol (100 mL). The cartridge was reconditioned and the remaining 13 samples were washed and extracted as previously described. The organic solutions were combined and rotary evaporated under reduced pressure. The residue was redissolved in DI water and lyophilized using a Labconco freeze dryer (100×103 mbar, −40° C.). Sensory analysis confirmed the presence of the savory attribute in the organic fraction.
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Acetone
Aluminum
Amniotic Fluid
ARID1A protein, human
butyl acetate
Cacao
Centrifugation
Cocoa Powder
Dietary Fiber
Ethanol
ethyl acetate
Ethyl Ether
Flavor Enhancers
Fractionation, Chemical
Freezing
Glucosides
High-Performance Liquid Chromatographies
HMGB Proteins
Hydrolysis
Methanol
methyl acetate
Methylene Chloride
Neck
Nitrogen
Pellets, Drug
Polytetrafluoroethylene
Powder
Pressure
Procyanidins
Rubber
Salts
Savory
Solid Phase Extraction
Solvents
Syringes
Taste
Thermometers
Vacuum
West African People
Xanthines
Sodium borohydride, sodium chloride, Dowex cation-exchange resin (50W-X8), ammonium bicarbonate (ABC), TFA, Dulbecco’s PBS (DPBS), hydrochloric acid (HCl), and dl -DTT were purchased from Sigma–Aldrich. Ethanol (Reag. Ph. Eur) and bovine submaxillary mucin (BSM), type I-S, were purchased from Merck. Tandem mass tag (TMT)pro label reagents, 8 M guanidine hydrochloride, Dulbecco’s modified Eagle’s medium, 0.25% trypsin/EDTA, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. Potassium hydroxide was obtained from Honeywell Fluka. Solid phase extraction bulk sorbent carbograph was obtained from Grace Discovery Sciences. HPLC SupraGradient acetonitrile (methyl cyanide [MeCN]) was obtained from Biosolve. Peptide N-glycosidase F (PNGase F) and complete EDTA-free protease inhibitor cocktail tablets were purchased from Roche Diagnostics. A 96-well PP filter plate was purchased from Orochem Technologies. MultiScreen HTS 96 multiwell plates (hydrophobic Immobilon-P polyvinylidene difluoride [PVDF] membrane) and 96-well PP microplate were obtained from Millipore.
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acetonitrile
Acetonitriles
ammonium bicarbonate
BSM1 protein, Bos taurus
Cation Exchange Resins
Diagnosis
Dietary Fiber
Dowex
Eagle
Edetic Acid
Endo-beta-N-Acetylglucosaminidase F
Ethanol
Fetal Bovine Serum
High-Performance Liquid Chromatographies
Hydrochloric acid
Hydrochloride, Guanidine
Immobilon P
polyvinylidene fluoride
potassium hydroxide
Protease Inhibitors
sodium borohydride
Sodium Chloride
Solid Phase Extraction
Tissue, Membrane
Trypsin
Three biological replicates from each time point were analyzed. Frozen cell pellets containing ∼2.0 × 106 cells were resuspended in 100 μl of lysis buffer containing Tris–HCl, EDTA, sodium chloride, and protease inhibitor cocktail. The cells were lysed using a Branson sonicator rod at 1.5 output power, and 25 μl of the suspension (containing ∼5 × 105 cells) were loaded onto the preconditioned PVDF membrane plate wells. BSM (10 μg) was blotted in three different wells of the same PVDF membrane plate. The denaturation as well as the N- and O-glycan release were performed as described previously (31 (link), 33 (link)). Briefly, the proteins were denatured on membrane using guanidine hydrochloride and DTT at 60 °C. Upon removal of denaturing agents, the N-glycans were released by PNGase F digestion overnight at 37 °C and recovered in MQ water. A total of 2 units of PNGase F was added to each well of the PVDF membrane plate containing lysates from approximately 0.5 million cells. Upon recovery of the N-glycans, the O-glycans were released from the same wells by reductive β-elimination, using 50 μl of 0.5 M sodium borohydride in 50 mM potassium hydroxide incubating at 50 °C for 16 h. Samples were desalted by performing Dowex cation exchange resin (50W-X8) and graphitized carbon solid phase extraction in self-packed 96-well filter plates. The samples were dried after cleanup and stored at −20 °C until analysis.
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Biopharmaceuticals
Carbon
Cation Exchange Resins
Cells
Digestion
Dowex
Edetic Acid
Freezing
Glycopeptidase F
Hydrochloride, Guanidine
Pellets, Drug
Polysaccharides
polyvinylidene fluoride
potassium hydroxide
Protease Inhibitors
Proteins
sodium borohydride
Sodium Chloride
Solid Phase Extraction
Tissue, Membrane
Tromethamine
Release of N-glycan, Rapiflour labeling and purification of Rapiflour labeled N-glycan were performed according to the manufactures’ protocols. Briefly, 15 µg of each enzyme was heat denatured at 90°C for 3 min in 6 µL of buffer solution containing 5% (w/v) RapiGest SF and 18.2 megohm water. After cooling down to room temperature enzymes were deglycosylated with 1.2 µL of RapiPNGase F at 50°C for 5 min. Thereafter, the enzymes were labeled with 12 µL of the RapiFlour-MS Reagent Solution at room temperature for 5 min and 358 µL of acetonitrile solution was added to dilute the reaction. To enrich the glycans, hydrophilic interaction liquid chromatography solid phase extraction (HILIC SPE) was performed by Waters GlycoWorks HILIC µElution Plate. The plate was washed with 200 µL of Milli-Q water, followed by equilibration with 200 µL of 15:85 water/acetonitrile. After loading the acetonitrile-diluted sample, the well was washed twice with 600 µL of 1:9:90 (v/v/v) formic acid/water/acetonitrile. The glycans were eluted with 30 µL of GlycoWorks SPE Elution Buffer (200 mM ammonium acetate in 5% acetonitrile).
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acetonitrile
ammonium acetate
Buffers
Enzymes
formic acid
Hydrophilic Interactions
Liquid Chromatography
Polysaccharides
Solid Phase Extraction
Extraction of liver lipids was performed according to the method described by Folch et al.30 (link). Dihydroceramides and ceramides and were isolated by solid phase extraction chromatography using C12:0 dihydroceramide and C17:0 ceramide (Avanti Polar Lipids, Alabaster, Al, USA) as internal standards. Samples were analysed by liquid chromatography-mass spectrometry (LC–MS) using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempsted, UK) equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 ultra-high-pressure liquid chromatography (UHPLC) system. Samples were injected onto a Thermo Hypersil Gold C18 column (2.1 mm by 100 mm; 1.9 μm) maintained at 50 °C. Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid. Mobile phase B consisted of a 90:10 mixture of isopropanol-acetonitrile containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid. The initial conditions for analysis were 65% mobile phase A, 35% mobile phase B and the percentage of mobile phase B was increased from 35 to 65% over 4 min, followed by 65% to 100% over 15 min, with a hold for 2 min before reequilibration to the starting conditions over 6 min. The flow rate was 400 μl/min and samples were analyzed in positive ion mode. The LC–MS data were processed with Thermo Xcalibur v2.1 (Thermo Scientific) with signals corresponding to the accurate mass-to-charge ratio (m/z) values for dihydroceramide and ceramide molecular species extracted from raw data sets with the mass error set to 5 ppm. Quantification was achieved by relating the peak area of the dihydroceramide and ceramide lipid species to the peak area of their respective internal standard. All values were normalised to the wet weight of liver.
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acetonitrile
Alabaster
Altretamine
Ceramides
Chromatography
dihydroceramide
formic acid
formic acid, ammonium salt
Gold
High-Performance Liquid Chromatographies
Isopropyl Alcohol
Lipids
Liquid Chromatography
Liver
Mass Spectrometry
Solid Phase Extraction
Top products related to «Solid Phase Extraction»
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Trypsin is a serine protease enzyme that is commonly used in cell culture and molecular biology applications. It functions by cleaving peptide bonds at the carboxyl side of arginine and lysine residues, which facilitates the dissociation of adherent cells from cell culture surfaces and the digestion of proteins.
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Formic acid is a colorless, pungent-smelling liquid chemical compound. It is the simplest carboxylic acid, with the chemical formula HCOOH. Formic acid is widely used in various industrial and laboratory applications.
Sourced in United States, Ireland
Sep-Pak is a solid-phase extraction (SPE) device produced by Waters Corporation. It is designed to extract, purify, and concentrate analytes from liquid samples prior to analysis. The Sep-Pak device contains a sorbent material, which selectively retains the target analytes, allowing for the removal of unwanted matrix components. This process helps to improve the sensitivity and accuracy of subsequent analytical techniques.
Sourced in United States, Germany, United Kingdom, Japan, France, Canada, Ireland, Morocco
Oasis HLB cartridges are solid-phase extraction (SPE) devices used for sample preparation in analytical chemistry. They are designed to capture and concentrate analytes of interest from liquid samples. The cartridges contain a polymeric sorbent with both hydrophilic and lipophilic (hydrophobic) characteristics, which enables the selective retention of a wide range of polar and non-polar compounds.
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The Oasis HLB is a solid-phase extraction (SPE) cartridge developed by Waters Corporation. It is designed for sample preparation and cleanup prior to chromatographic analysis. The Oasis HLB cartridge utilizes a balanced-hydrophilic-lipophilic-balanced (HLB) sorbent to provide efficient extraction and recovery of a wide range of polar and non-polar analytes from various sample matrices.
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Methanol is a clear, colorless, and flammable liquid that is widely used in various industrial and laboratory applications. It serves as a solvent, fuel, and chemical intermediate. Methanol has a simple chemical formula of CH3OH and a boiling point of 64.7°C. It is a versatile compound that is widely used in the production of other chemicals, as well as in the fuel industry.
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Acetonitrile is a colorless, volatile, flammable liquid. It is a commonly used solvent in various analytical and chemical applications, including liquid chromatography, gas chromatography, and other laboratory procedures. Acetonitrile is known for its high polarity and ability to dissolve a wide range of organic compounds.
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Methanol is a colorless, volatile, and flammable liquid chemical compound. It is commonly used as a solvent, fuel, and feedstock in various industrial processes.
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Iodoacetamide is a chemical compound commonly used in biochemistry and molecular biology laboratories. It is a reactive compound that selectively modifies cysteine residues in proteins, thereby allowing for the study of protein structure and function. Iodoacetamide is often used in sample preparation procedures for mass spectrometry and other analytical techniques.
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Formic acid is a clear, colorless liquid chemical compound used in various industrial and laboratory applications. It is the simplest carboxylic acid, with the chemical formula HCOOH. Formic acid has a pungent odor and is highly corrosive. It is commonly used as a preservative, pH adjuster, and analytical reagent in laboratory settings.
More about "Solid Phase Extraction"
solid-phase sample preparation, solid-phase extraction, spe, trypsin, formic acid, sep-pak, oasis hlb, oasis hlb cartridges, methanol, acetonitrile, iodoacetamide, analyte purification, analyte concentration, analyte isolation, sample clean-up, sample preparation, analytical chemistry, biochemistry, proteomics, lc-ms, liquid chromatography-mass spectrometry, reproducibility, sensitivity, optimization, ai-powered solutions, pubcompare.ai