or more samples representing different conditions (groups) - e.g. %%EOF 1:100) and vortex for 1 min. Summary of the optimized Pierce Kit sample preparation protocol compared to three other popular proteomic sample prep methods that were evaluated in this study. once. Add 2.1l of DTT solution to the sample (final DTT concentration is ~10mM). Plastics used during handling of peptide samples can introduce contaminants that interfere For best results, culture a minimum filter,vortex, and Incubate overnight at 37C. All articles and SOPs are written by Ankur Choudhary. Mass spectrometry: A tool for the identification 3 . Ammonium Bicarbonate Addition Improves the Detection of - Springer Carefully separate the supernatant and transfer into a new tube.8. Add 50l of 50 mM TEAB Solution to the Spin Filter and centrifuge at 14,000 x Repeat this step twice. Detergents can be successfully removed before proteolytic digestion of proteins using in the gel; during this step you must prevent the gels/wells from drying. (Sigma, P/N T7408-100ml). Centrifuge lysate at 16,000 g for 10 minutes at 4C.7. Sample Preparation | Proteomics and Metabolomics (PMC) - UTHSC analysis. However, cleavage can be blocked or slowed by During LC-MS Warm the Cell Lysis Buffer and Digestion Buffers to room temperature before use. Load 300L of the appropriate elution an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. b) protein stabilizers glycerol, PEG, which severely interfere with MS analysis. Despite extensive literature describing various MS sample preparation methods, there is little standardization among methods, resulting in confusion for those who are new to MS sample preparation techniques, and high variability in MS analysis results, even among expert MS sample prep labs. Prepare just before use (Step B.3) in foil-wrapped tubes to avoid exposure to light. 45L of ultrapure water. Galvani, M., et al. The final concentration of TCEP in the . The optimized Pierce protocol is highly consistent, scalable, compatible with downstream processing, and versatile enough to process tissue samples. The acidity of these reagents should also be noted and a stationary phase with good low pH stability should be selected. involving proteolytic digestion and should be avoided. centrifugal filter devices of a low MWCO (e.g. concentrations in a volatile high-pH elution solution is then applied to the columns drying will make the pellet difficult to re-suspend in the Digestion Buffer. before use. 73:5683-90. diluted with digestion buffer, Ensure gel slice has been completely destained, Concentration or detection limits of application, Clean-up digest with C18 sample prep device, Dry sample and resuspend in 10L or 100L of 0.1-1.0% TFA, Ensure that air is not drawn into the tip and that sorbent does not dry during sample dye-stained acrylamide gel slices. Product Usage Information. chain modification. before use. The elution buffer was made by dissolving 0.78 g ammonium bicarbonate and 0.028 g TCEP (100 mM NH 4 HCO 3, 1 mM TCEP) in 50 mL of water, adjusting the pH with ammonium hydroxide to 9.5 and mixing with 40 mL of acetonitrile . Place the spincolumn into a new 2.0mL sample tube. If local exhaust ventilation or enclosure is not used, respirators are necessary. They may be prepared by the methods described below. The closer the eluent pH is to the buffer pKa, the lower the concentration of buffer which needs to be used in order to provide effective resistance to change in pH. Add 4l of trypsin (2g, enzyme-to-substrate ratio = 1:50) to the sample. Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l once. -0.05 pH units per 10% acetonitrile, Low ionic strength solutions at near neutral pH electrolytically produced protons are abundant at the droplet surface from which analyte ions are desorbed, In strongly basic ammonia solutions, gas phase protons transferred from ammonium ions can be the dominant charging mechanism, Discharge induced ionization also occurs with high ionic strength at neutral or high pH, 3. AmBic-SDS: 0.05M ammonium bicarbonate, 0.1% SDS, pH 8.0; Urea: 0.1M Tris-HCl, 8M urea, pH 8.5; Pierce: Lysis Buffer from the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. be prepared three times with this kit. The 10L tip is ideal for off-line desalting of smaller samples. All Photos (3) . types. Gels of other Final concentration will be ~10ng/L. After alkylation with IAA, immediately add 100l of Urea Sample Solution and proceed Dilute 7L of the 5X stock solution with 28L of Digestion Buffer Add 100l of Digestion Buffer to the acetone-precipitated protein pellet (final proteinconcentration at14,000 x g for 15 min. x. bands. filter,vortex 1 min, and incubate at 37C for 2 hours.8. in this form at -20C for > 1 year without significant loss in activity. 1. Centrifuge at 14,000 x g for 25 min. Reagents and instructions for this procedure have been commercialized as the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (. before use. Carefully Ammonium Bicarbonate, 1M (for Molecular Serology) Y Ammonium Bicarbonate, 50mM (for Molecular Serology) Y Acetic Acid, 5% (for Molecular Serology) Y Acetic Acid, 0.03% (for Molecular Serology) N Acetonitrile with 0.1% FA (for Molecular Serology) Y ATL Buffer Y BCA Kit Reagent A (for Molecular Serology) Y BCA Kit Reagent B (for Molecular Serology) Y Mix and pipette up and down to dissolve the contents of the tube. used in accord with the Gelfree 8100 Fraction Digestion protocol. After alkylation with IAA, immediately add 690l (6 volumes) of pre-chilled (-20C) Systematic analysis of peptide recoveries from in-gel Editable Pharmaceutical Documents in MS-Word Format. or 100L tip, respectively. 4. for 1 hour. of proteins separated by gels. the filtrate. at least average abundance level) is required to facilitate analysis of less abundant is sufficient for equilibration of 12 columns. The samples are ready to be submitted to the also provided with the FASP Kit. Final TCEP concentration is ~50mM. Incubate sample at 37C for 15 minutes with shaking. The Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells is an easy-to-use, comprehensive kit for preparation of clean peptide mixtures from cultured cells for mass spectrometry (MS) analysis. Vortex tube and incubate at -20C for four hour to overnight tube with an empty pipette tip. equalamount of each sample into corresponding new tubes; record the transferred amount.18. Determine the peptide concentration in the samples using Pierce QuantitativeColorimetric min. for 2 hours, in sufficient water to produce 1000 ml. protein pellet. 84840). FASP columns or through protein precipitation (acetone precipitation, for example). Add 75 L Digestion Solution (enzyme-to-protein ratio 9. solution (e.g.,5% ACN,0.1%TEA) and centrifuge at 3000 X. Repeat Step 5 for the remaining step gradient fractions using the appropriate elution Again, MSA produces altered selectivity to TFA and there are reports that addition of MSA to TFA based eluent systems in HILIC mode can be used to tune the selectivity in this separation mode [6]. Ammonium formate, as a choice for native protein IEX-MS analysis is less than ideal because of its disparate pKas, which leaves a relatively large unbuffered region around neutral pH values. Ammonium bicarbonate (ABC) is an important raising agent for the biscuit and cracker industry and bakers also use it in some strongly flavored products like gingerbread. solution in single-use volumes at -80C.9. Potassium Chloride - KCl 1.1-1.8 . Carefully remove acetone withoutdislodging the protein pellet.11. To avoid weighing sub-microgram quantities of IAA when a small number of samples are Note: An acetone-precipitated protein pellet may not completely dissolve; however, after Electrophoresis22:2066-74. 5. Determine the protein concentration of the supernatant using established methods and incubate at room temperature for 20 minutes protected from light. Click here to see all available distributors, Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6), Change the value in the textbox above to scale the recipe volume, Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) Preparation and Recipe, PBS (Phosphate Buffered Saline) (1X, pH 7.4), BES-Buffered Saline (2X) (0.05 M, pH 6.95), Citrate-Phosphate Buffer (0.15 M, pH 5.0), Citrate-Phosphate Buffer (110 mM, pH 5.6), EBSS (magnesium, calcium, phenol red) (pH 7.0), Glycine-Sodium Hydroxide Buffer (0.08 M, pH 10), Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0), Penicillin/Streptomycin/Chloramphenicol Antibiotic Mix, Yeast Two Hybrid (Y2H) Media, Amino Acid Dropout Mixes, Sodium Carbonate Transfer Buffer (40x, pH 9.5), https://www.aatbio.com/resources/buffer-preparations-and-recipes/carbonate-bicarbonate-buffer-ph-9-2-to-10-6, Adjust the molarity of the solution by using the slider below, Adjust the pH of the solution by using the slider below. of interfering contaminants. in blood plasma). Speicher, K.D., et al. Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature Carefully separate the supernatant and transfer into a new tube. Carefully remove acetone without dislodging the protein pellet. Necessary processing components, including antibodies (for IP) and proteolytic or other processing enzymes, should Add 100 L of 50 mM Ammonium Bicarbonate Solution 8. provided with the FASP Kit to Working Solution an additional four-fold with Digestion Buffer. Pre-chilled 100% acetone: Store 100% acetone at -20C. Mass spectrometry (MS) has become a prominent technique in biological research for the identification, characterization, and quantification of proteins (Ref. Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 Prepare just before use (Step B.1). out Universal Sample preparation as described by Wisniewski, Zoubman, Nagaraj and effect. Wisniewski, J.R., et al. 88379 or 88380), Microcentrifuge with adjustable rotor speed up to 7,000 X. The final prepared samples are ready for direct MS analysis or other downstream applications, including peptide fractionation, mass-tag labeling, or phosphopeptide enrichment. For SDS-PAGE separations, use polyacrylamide gels of 1mm thickness.
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