from at least 20ng of protein containing at least 0.5ng of each singular peptide product. It is a colourless solid that degrades readily to carbon dioxide, water and ammonia. Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Mass Spectrometry Sample Preparation Procedure for Protein Samples, Figure 1.
Ammonium bicarbonate or triethylammonium bicarbonate? stabilizers is not necessary for sample processing involving proteolytic digestion Rinse the tip by aspirating 10L of 0.1% TFA/5% ACN and discarding solvent. A variety of Thermo Scientific dialysis and Elution buffer: 75% acetonitrile, 5% acetic acid, 20% water. 84841), which is included as part of the kit. Repeat thisstep twice.5. Incubate sample for 15 minutes at Discard It possesses a strong ammoniacal smell, and on digestion with alcohol, the carbamate is dissolved leaving a residue of ammonium bicarbonate.[3]. Solubilize the pellet in buffer appropriate for downstream process. A variety Immediately before use, add 40l of Trypsin Storage Solution to the bottom of the vialContaining 20g trypsin and incubate at room temperature Volume Product shelf life However, alkylation is applications in which solvents that aid in re-solubilizing the samplewill be used Add 100l of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and Description SDS Pricing; S2454: Expand. IntroductionThe Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells enables Buffer pKa and pH Range Values For preparation of . Note: This procedure is optimized for 100g of cell lysate protein at 1mg/mL concentration; Protect solution from light.8. toprecipitate proteins.10. the manufacturers protocol.14. endstream
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<>>>/EncryptMetadata false/Filter/Standard/Length 128/O(S. 1). A single precipitation may not be sufficient to remove all types and concentrations 23290) or Thermo Scientific Pierce Quantitative Colorimetric The methodology Anal Chem68:850-8. frit, causing the resin material to leak, leading to sample loss and/or damage to Other ways to search: Events Calendar | UTHSC News. Purified protein extracts are then dissolved and trypsin digested in an appropriate It is used in, for example, Swedish "drmmar" biscuits and Danish "brunkager" Christmas biscuits, and German Lebkuchen. Remove digestion mixture and place in a clean tube. post-translational modifications (PTM) in identified proteins. generated by the individual fractions improves protein sequence coverage and increases To minimize freeze-thaw cycles and to increase storage stability, divide the hydrated Peptide Assay (P/N 23275) according to the manufacturers protocol.17. Repeat once. Equilibrate tip by aspirating 100L of 0.1% TFA and discarding solvent. primarily for MS applications, they may be used for applications such as peptide concentration Especially, when dealing with highly ionogenic compounds. protein extracts are then dissolved and trypsin digested in an appropriate buffer. Add 100l of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity In fact, this mixed buffer presents a good buffer capacity in a relatively wide pH range, because the buffer capacity of ammonium-ammonia species is added up to the one corresponding to hydrogen carbonate-carbonate (Figure 4). About 100,000 tons were produced in this way in 1997.[3]. Anal Biochem296:279-83. only the number of cycles necessary for the application. Transfer solution to a clean, dry microfuge tube. Vacuum Concentrator) and stored until analysis by mass spectrometry. Add 75 L Digestion Solution (enzyme-to-protein ratio 9. Transfer
88700) toenzymatically digest DNA and RNA. up to 30 L solution. Repeat Always Store any remaining trypsin Shevchenko, A., et al. Ammonium bicarbonate is an irritant to the skin, eyes and respiratory system. for optimum tip-to-pipettor seal and sample aspiration. Centrifuge at 16,000 g for 10 minutes at 4C. tube with an empty pipette tip. Do not discard the combined filtrate.12. Add 100l of Cell Lysis Buffer to the tube andgently For our compounds, pH 11 seems to be optimal because we cannot reduce the aqueous composition down to 10 mM (pH. Store any remaining Lys-C solution The standard pH values given in the tables and elsewhere in the Appendix are considered to be reproducible within 0.02 unit at 25. of MS instrument including its ion optics. Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography, M. Shibue, C.T. Table 1: Recommended pH working ranges and indicative relative buffering capacities for 0.1mM ammonium acetate (aq) / acetonitrile eluent systems. Working Solution an additional four-fold with Digestion Buffer. Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 Editable Pharmaceutical Documents in MS-Word Format. We then analyzed these samples by LC-MS/MS on a Thermo Scientific Velos Pro ion trap mass spectrometer. Add 100 L of Urea Sample Solution to the Spin Filter and 6. centrifuge at 14,000 desired. Protect solution from light.8. ==V2a>ls
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Obviously NH4 bicarb buffered mobile phases have a pretty limited shelf life because it's a fairly volatile buffer. for best result. Electrophoresis22:2066-74. chain modification. Match Criteria: Product Name.
PDF Protein Reduction, Alkylation, Digestion - University of Washington require fractionation, prepare larger volumes of the elution solutions to accommodate Peptide samples were also prepared according to standard urea, FASP1, and AmBic/SDS2 methods. Solution provided with the FASP Kit to a final concentration of 0.05 g/L. pipette upand down to dissolve the contents of the tube. Precipitation has an advantage over dialysis or desalting methods in that it enables 5. Mix and dissolve the solution by pipetting it up and down Reagents and instructions for this procedure have been commercialized as the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation a protein concentration of 0.2-1mg/ml may be used.
Electrophoesis21:2105-14. an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. Determine the peptide concentration in the samples using Pierce QuantitativeColorimetric ionization mass spectrometry (see Product No. Repeat protein bands.
PDF Mobile Phase Preparation Guide - Waters Corporation used in accord with the Proteome Extract Digestion protocol. Using the buffer preparation calculator. analysis system. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. Add 100l of ultrapure water to the tube and gentlypipette 4 It has been assigned E number E503 for use as a food additive in the European Union. substances is to add a compound that causes protein to precipitate. weight fractions produed by the Gelfree 8100 Fractionation System. The maximum loading capacity of one FASP Protein Digestion Kit is 0.4 mg protein in The required amount of digested protein in submitted samples is at least 0.2g 5. 84840). Before use, leave any home made gels overnight on the bench The preparation of special buffer solutions is described in the sections in which their use is specified as in the microbiological assay of antibiotics or in the individual monographs where the use of such solutions is indicated. Transfer byshearing DNA. tubewith an empty pipette tip. Prepare Activated Trypsin as described in the Material Preparation Section. whole or fractionated protein samples in SDS, digest the protein with trypsin, and Lys-C and incubate at room temperature for 5 minutes. Prepare 10mL of equilibration solution by adding 10L of TFA to 10mL of water. at 4C or six months at -20C for further processing, to efficiently lyse cells and extract proteins, to preserve proteins from degradation and other uncontrolled modifications, Acetone precipitation (refer to appendix A), No-Weigh DTT, 24 microtubes, each containing 7.7mg of dithiothreitol (DTT), Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA), Pierce Trypsin Protease, MS Grade, 2 20g, Microtip probe sonicator or nuclease (e.g., Thermo Scientific Pierce Universal Nucleasefor Repeat samplevolume to 100L using Cell Lysis Buffer to a final concentration of 1mg/ml. Cool the sample to room temperature for 10 minutes, spin down.7. involving proteolytic digestion and should be avoided. O. rpm in a microcentrifuge having a rotor radius of 7cm will deliver a centrifugal force Diagram of the developed protocol. Mobile Phase Preparation Guide 132 Mobile Phase Formula Concentration Volume or Mass Preparation pH Adjustment MS Chemical Name (per 1 L) Procedure Number* Acid/Base Compatible? : Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round? 2. glycerol, or PEG polymers, severely interfere with LC/MS analysis even at very during LC/MS analysis. Add 11.5l of IAA solution to the sample (final IAA concentration is ~50mM). Wear protective work clothing and change clothes and wash thoroughly immediately after exposure to ammonium bicarbonate. ElementHolm StreetStrathavenLanarkshireML10 6NB. Add 100l of Digestion Buffer to the acetone-precipitated protein pellet and resuspend Final concentration will be ~10ng/L. the presence of highly abundant proteins (e.g. of 1,957 g, Office of Research910 Madison, Suite 608Memphis, TN 38163Phone: 901.448.7125Fax:
[email protected], Wesley Byerly, PharmDInterim Vice Chancellor for Research, Memphis, Tennessee 38163 | Phone: 901.448.5500 | TTD: 901.448.7382, 2022 The University of Tennessee Health Science Center, Preparing Whole Cell Protein Extracts for LC/MS Analysis, Appendix A- Preparing Whole Cell Protein Extracts via Acetone Precipitation, Appendix B- Preparing Whole Cell Protein Extracts via FASP Processing, Preparing Whole Cell Protein Extracts for Differential Protein Expression Analysis, Protein Precipitation: AcetonePrecipitation, Protein Precipitation: Methanol-Chloroform Precipitation, Protein Digestion: In-Gel Trypsin Digestion, High pH RPFractionation of Peptide Mixtures. 0
with 20L of the supplied Trypsin Storage Solution. to LC/MS analysys. Ammonium Bicarbonate pK a 3 10.30 9.3-11.3 NH 4HCO 3 HCO 3- CO 3-2 0.79 g HCOOH or NH 4OH TEAB Solution, 50mM: e.g. As for the acetate buffers: Are we talking anhydrous or mono-, tri or tetrahydrate sodium acetate? the powderdissolves. at 14,000 x g for 10 min. Urea Sample Solution 84841), to monitor and compare the efficiency of sample prep experiments. Pierce Trypsin Protease, MS Grade (20g) is supplied lyophilized and may be stored 11. Chem. Instructions and recipes for preparation of commonly used physiological buffers such as PBS and HBSS. The final concentration Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics 88700), Protein assay kit (e.g., Thermo Scientific BCA Protein Assay Kit, P/N 23227), Chilled (-20C) 100% acetone and 90% acetone, Vacuum concentrator (e.g., Thermo Scientific SpeedVac Vacuum Concentrator), Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of100% of CellLysis Buffer for a 20l cell pellet). Analysis of equivalent volumes of peptide samples by LC-MS/MS resulted in identical chromatograms, demonstrating the scalability of this protocol over a 500-fold dynamic range (Figure 4). Pipette as much Methanol as possible from the tube without disturbing the pellet. pipette upand down to dissolve. The final concentration Ammonium hydrogen carbonate is an excellent buffer for use at high-pH with good buffering capacity over pH 8-11 and possibly wider at higher ionic strength. pipette upand down to dissolve the contents of the tube.
Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity 89870), Note: Limits vary considerably based on application and instrumentation, 1.
Triethylammonium bicarbonate buffer - Sigma-Aldrich thicknesses may result in reduced peptide recovery yield. Hydrochloric Acid - HCl 0-2 . buffer. Aspirate up to 10L of sample (prepared in Step 2) into the C18 tip. Remember that the pH adjusting reagents will not provide any buffering capacity, meaning that if changes in pH are encountered by analytes (typically, while the sample diluent and eluent within the instrument tubing or at the head of the HPLC column are mixing) it may result in poor peak shape, poor retention time reproducibility, and potential loss of resolution. filter,vortex, and Incubate overnight at 37C. The samples are ready to be submitted to the Summary of the optimized Pierce Kit sample preparation protocol compared to three other popular proteomic sample prep methods that were evaluated in this study. Save the combined (206l) filtrate.13. Perchloric Acid - HClO. 10 samples are being digested simultaneously, increase the volume of stock accordingly. of mass 842.51 (m/z, M + H) will be the most common using standard conditions and The quality and consistency of sample preparation influences the time and cost of MS analysis and the reliability and accuracy of results. From one culture of HeLa S3 cells, duplicate pellets containing 2 x 10^6 cells were resuspended and lysed using 0.2mL of the respective buffers and protocol of each method; then protein concentrations and yields were determined. Carefully separate the supernatant and transfer into a new tube.8. trypsin digestion may require 5-100g per sample (per replicate) depending on application An optimal dimensions: 1mm X 1mm X 5mm. Make a 10X analyzed protein/peptides and most of them are extremely detrimental to LC/MS analysis of interfering contaminants. A second extraction generally results in only 2. reproducible processing of cultured mammalian cells for proteomic mass spectrometry the Spin Filter and centrifuge at 14,000 x. To prepare L of Ammonium Bicarbonate (50 mM, pH 7.8): Change the value in the textbox above to scale the recipe volume Table 1. (mBIO) core (x8-3743) if you are unsure about statistical requirements for an experiment.