Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the Approx. Warm the Cell Lysis Buffer and Digestion Buffers provided with Pierce kit to roomtemperature be prepared three times with this kit. Solubilize the pellet in buffer appropriate for downstream process. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the Excess IAA has been supplied with this kit. Wash buffer: 0.1% acetic acid in water. protein extracts are then dissolved and trypsin digested in an appropriate buffer. at 14,000 x g for 10 min. One might expect that selecting an eluent pH in which the analyte is expected to be in the neutral form (eluent pH above the analyte pKa for basic analytes) will lead to reduced analyte detector response. Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) Preparation and Recipe During LC-MS Plastics used during handling of peptide samples can introduce contaminants that interfere Add 4l of trypsin (2g, enzyme-to-substrate ratio = 1:50) to the sample. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge g for 10min. Rinse the tip by aspirating 100L of 0.1% TFA/5% ACN and discarding solvent. Repeat this step once. These pH adjusting reagents and buffer combinations are shown in Table 1. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. Standard buffer solutions for various ranges of pH values 1.2 to 10.0 may be prepared by appropriate combinations of 0.2 M hydrochloric acid or 0.2 M sodium hydroxide and of solutions described below, used in the proportions shown in the accompanying tables. at room temperature for 15 minutes. To avoid weighing sub-microgram quantities of IAA when a small number of samples are Comments shall be published after review. for 5 minutes. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge Transfer the Spin Filter to a new collection tube. When using 10g of cell lysate, : Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round? before use. 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]. 5 The unbuffered region leads to unoptimized separations and irreproducible elution. ~25mM). Add 10 L 10X Iodoacetamide Solution and 90 L Urea 4. 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. theSpin Filter at 14,000 x g for 10 min. Vortex the tube until all The final concentration trypsin). x. Transfer the Spin Filter to a new collection tube. solution in single-use volumes at -80C.9. Peptide Assay (P/N 23275) according to the manufacturers protocol.17. Incubate the lysate at 95C for 5 minutes.4. Table 1. Wrap the tops of the tubes with Parafilm Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l also provided with the FASP Kit. settings for your system, Verify LC-MS system performance with the Thermo Scientific Pierce HeLa Digest Protein Centrifuge Carefully remove acetone withoutdislodging Mix 3.3L of TCEP with 30L of Digestion Buffer The Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit provides Add 100l of Cell Lysis Buffer to the tube andgently Carefully toSection D, FASP Protein digestion. Detergents are usually difficult to remove from digested protein samples Adjust the pH to 3.7 with 10 M ammonia and dilute with water to 100 ml. However, cleavage can be blocked or slowed by anddesalt using C18 ZipTips (or equivalent) of appropriate capacity according to concentrate digest on C18 sample prep device (Product No. 89870), Note: Limits vary considerably based on application and instrumentation, 1. Ammonium bicarbonate - Wikipedia Final concentration will be ~10ng/L. [ 1] [ 2] Ammonium bicarbonate buffer regulates . plan accordingly. A second protocol, included, provides instructions for digesting molecular Pipette as much Methanol as possible from the tube without disturbing the pellet. This Pierce procedure incorporates two-stage enzymatic digestion with LysC and trypsin proteases. if your samples are degraded or contain a particular protein at high abundance that (per replicate). . inhibited or slowed by a variety of conditions, such as the presence of thiourea, Add 100l of ultrapure water to thetube and gentlypipette Figure 4. P/N 23227). Protein extracts can be separated from these low MW components by filtration using Transfer Several methods for protein precipitation are described in the literature. There is no absolute single best way to lyse cells and extract proteins. or stabilizers such as glycerol, or PEG polymers. Prepare Alkylation Buffer as described in the Material Preparation Section. 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. Well, this procedure is good enough for a rough screening solution- call it a quick and dirty method. Anyone know how to prepare 0.2 M bicarbonate. Store the solution in tightly sealed bottles at 4C or at room temperature. This method yields more protein lysate from cultured cells, is highly reproducible, is scalable from 10g to 5mg, is simpler and faster than FASP, has no risk of carbamylation by urea, and results in higher protein identification rates than other popular standard sample preparation methods (Figure 2 and Table 2). Store any remaining Lys-C solution Wrap the tops of the tubes withParafilm Do not exceed the recommended centrifugation speeds because this may damage the column Note:Do not store the Alkylation Buffer or stock solution. It is a colourless solid that degrades readily to carbon dioxide, water and ammonia. Note: Do not dry the acetone-precipitated protein pellet for more than 2-3 minutes; excess How do I prepare pH 11 Ammonium Bicarbonate Buffer for LCMS? Evaluation of the efficiency of in-gel is removed and theprotein pellet is re-dissolved in a buffer that is compatible with is sufficient for equilibration of 12 columns. Ammonium acetate buffers - Crawford Scientific of trypsin can be reliably used for a wide variety of protein concentration within Alternative destaining procedures are required for silver- or zinc-stained However, because some sample loss will accompany each cycle ofprecipitation, use Spectroscopy, Elemental and Isotope Analysis, Thermo Scientific Pierce Mass Spec Sample Prep Kit, Remove SDS by urea washes and spin concentrator, Recover peptides by NaCl washes and spin concentrator, Digestion indicator sequence coverage (%), FASP: 0.2mL of 0.1M Tris-HCl, 4% SDS, 0.1M DTT, pH 7.6, AmBic-SDS: 0.05M ammonium bicarbonate, 0.1% SDS, pH 8.0, Pierce: Lysis Buffer from the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (. NOTE: The 30 mg/mL TCEP stock solution must be prepared in 16 mg/mL (~200 mM) ammonium bicarbonate to bring up its pH. for ESI-MS, up to 100L per sample. tube with an empty pipette tip. 23227). Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of100% 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. However, we observed 20-25% missed cleavages when the same samples were analyzed on Thermo Scientific Q Exactive or Orbitrap Elite instruments. Buffer to the tube. 100%acetone to sample. Store any remaining Lys-C solution Place the spincolumn into a new 2.0mL sample tube. endstream endobj startxref 2) Is it. 19 0 obj <> endobj Mass spectrometry (MS) has become a prominent technique in biological research for the identification, characterization, and quantification of proteins (Ref. below). weight fractions produed by the Gelfree 8100 Fractionation System. 8. Solutions of sodium or ammonium acetate (for example) can be infused into the eluent flow post-column in order to promote adduct formation, which is often attendant with an increase in analyte signal. PDF Mobile Phase Preparation Guide - Waters Corporation Dissolve 4 g of anhydrous sodium acetate in about 840 ml of water, add sufficient glacial acetic acid to adjust the pH to 2.8 (about 155 ml) and dilute with water to 1000 ml. identified from complex samples by liquid chromatography-mass spectrometry (LC-MS) In suchcases, repeated precipitation may be performed. Comments having links would not be published. DMSO, DMF interfere with MS analysis. For maximum with 20L of the supplied Trypsin Storage Solution. This saves time and money when coming up against roadblocks with separation development as, once all of the usual buffers have been tried, attention turns to changing the column chemistry, which may not be necessary. Store buffers at 4C. sorbent that minimizes flow resistance and provides excellent binding and recovery the powderdissolves. This makes it extremely difficult for new MS users to find the best protocol and use it to obtain consistent results. X. . Hence, sensitivity of detection is not affected to the same degree as with TFA. 84841), to monitor and compare the efficiency of sample prep experiments. that inhibit trypsin digestion, and digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or nanospray Shevchenko, A., et al. From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by the Pierce protocol. Transfer Mix 5.3 ml of 0.2 M hydrochloric acid and 25 ml of 0.2 M potassium chloride, add 4 ml of a 0.393 percent w/v solution of cupric sulfate and dilute to. (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and Glycine Buffer Solution: Mix 42 g of sodium bicarbonate and 50 g of potassium bicarbonate with 180 ml of water and add a solution containing 37.5 g of glycine and IS ml of strong ammonia in 180 ml of water. Use a vacuum 15. concentrator to dry Prepare Activated Trypsin as described in the Material Preparation Section. filter,vortex 1 min, and incubate at 37C for 2 hours.8. Do not discard the combined filtrate.12. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity Filtrate contains digested protein fraction. post-translational modifications (PTM) in identified proteins. On the front-line of the selectivity battle, we need to have as many weapons as possible! Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity We carefully optimized the reaction buffers and protocol for high-resolution spectrometers to minimize non-selective alkylation or incomplete digestion. If using nuclease, add 25 units of nuclease Mix 80mg of ammonium bicarbonate with 20mL of acetonitrile (ACN) and 20mL of ultrapure to the hydrophobic resin under aqueous conditions and desalted by washing the column Prepare 800 mL of distilled water in a suitable container. (1996). at 4C. Unfortunately, when ammonium bicarbonate was used as a buffer reagent in electrospray ionization analysis, proteins formed higher charge states, indicative of protein denaturation . Remove and discard Destaining Solution from the tube. Wet tip by aspirating 100L of 50% ACN in water and then discarding solvent. Shotgun proteomics is a commonly used strategy to identify proteins in complex mixtures by digesting proteins at specific amino acids into peptides that can be separated and identified by mass spectrometry (Ref.2). onto the spin column, Increase vortexing/sonication time to completely dissolve the dried peptide sample, Incorrect centrifuge speeds used for fractionation, Low peptide/protein identification numbers, Estimate peptide concentration using the Thermo Scientific Pierce Quantitative Fluorometric The final concentration of DTT is~500mM. as 35% for hydrophilic peptides. Aftercentrifugation Urea Sample Solution. Match Criteria: Product Name, Keyword. the Spin Filter and centrifuge at 14,000 x. Place protein sample in acetone-compatible tube. Am. Make a 10X Salts, buffers, other small hydrophilic 0 In addition, Incubate sample at 37C for 30 minutes trypsin digestion may require 5-100g per sample (per replicate) depending on application Do not over-dry pellet, or itmay not dissolve properly. such asthe BCA Protein Assay Kit (e.g., Thermo Scientific BCA Protein AssayKit, frit, causing the resin material to leak, leading to sample loss and/or damage to gels. 14,000 x, Add 50 L 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge acetone with 5mL of ultrapure water) and store at -20C, Pre-chilled 100% acetone: Store 100% acetone at -20C. Buffer. in single-use volumes at -80C.7. Sample preparation can be performed in 2 alternative ways using, Microcentrifuge polypropylene tubesMicrotip probe sonicator or nuclease (e.g., Thermo Scientific Pierce Universal Nucleasefor Figure 2. a* Buffer Range Formula Buffering Equilibrium 10 mM Concentration Mobile-Phase Preparation** pH Adjustment (Acid or Base) Ammonium Acetate pK a 1 4.76 3.8-5.8 CH 3COONH 4 CH 3COOH CH 3COO-0.77 g CH . Urea Sample Solution If you have used Protein Discoverys UPX Universal Protein Extraction Kit or YPX 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 protein pellet. Buffer pKa and pH Range Values For preparation of . SpeedVac to dryness but avoid drying too long as this makes the pellet harder to 88379 or 88380), Microcentrifuge with adjustable rotor speed up to 7,000 X. Set the pipettor to 100L and secure the pipette tip tightly to the end of the pipettor 23225) or Thermo Scientific Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Part No. Olsson, I., et al. Seppro Ammonium Bicarbonate Buffer. b) protein stabilizers glycerol, PEG, which severely interfere with MS analysis. Place pieces into a 600L receiver tube. Store the remaining components It is used in, for example, Swedish "drmmar" biscuits and Danish "brunkager" Christmas biscuits, and German Lebkuchen. Pre-chilled 100% acetone: Store 100% acetone at -20C. peptide mixture samples can be fractionated using the kit. Retain eluate as wash fraction. at14,000 x g for 15 min. Determine the protein concentration of the supernatant using established methods such Each reversed-phase fractionation spin column Ammonium Acetate Preparation and Recipe | AAT Bioquest Remove extraction solution The required amount of digested protein in submitted samples is 25-100 g per sample Ammonium bicarbonate is produced by combining carbon dioxide and ammonia: Since ammonium bicarbonate is thermally unstable, the reaction solution is kept cold, which allows the precipitation of the product as white solid. for 5 minutes. Place 50.0 ml of 0.2 M potassium hydrogen phthalate in a 200 ml volumetric flask, add the specified volume of 0.2 M hydrochloric acid (see Table 2) and then add water to volume. Store in polyethylene containers. the Spin Filter at 14,000 x g for 10 min. of IAA is ~500mM. Resulting lysate samples (200g in 200L of Lysis Buffer) were spiked with 2g Digestion Indicator and processed through remaining steps of the Pierce protocol. The method also involves using an internal control-protein, called a Digestion Indicator (Part No. All the crystalline reagents except boric acid should be dried at 110 to 120C for 1 hour before use. Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics Screenshot of software analysis for indicator peptides. of peptide samples for desalting after digestion and before mass spectrometric analysis. Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT or more samples representing different conditions (groups) - e.g. Each cell suspension was sonicated on ice for 20 seconds (pulse time 5 sec, pulse off time 5 sec, output level 2) using a Misonix 3000 Sonicator.