Processing/preparation of protein extracts for LC/MS analysis include trypsin digestion. From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by each method. vialContaining 20g trypsin and incubate at room temperature for 5 minutes. For best results, culture a minimum fractionation, high-pH reversed-phase fractions do not require an additional desalting Some contaminants Effect of mobile phase additives on solute retention at low aqueous pH in hydrophilic interaction liquid chromatography, McCalley DV, Journal of Chromatography A, 1483 (2017) 71-79, 7. reducing agents dithiothreitol, beta-mercaptoethanol, and tris(2-carboxyethyl) phosphine. Gently pipette upand down to dissolve. silver stains or reversible zinc staining (Product No. P/N 23227), 5. such asthe BCA Protein Assay Kit (e.g., Thermo Scientific BCA Protein AssayKit, 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. Vortex tube and incubate for 60 minutes to overnight at -20C. phosphorylated, Thermo Scientific Tandem Mass Tag (TMT)-labeled, and other complex side of lysine and arginine residues. In-gel digestion coupled with mass spectrometric analysis is a powerful tool for the per 1ml ofcell lysate and incubate at room temperature for 15 minutes.6. We have developed an optimized protocol and kit of reagents that standardize peptide sample preparation for MS analysis (Figure 1). Buffer for each sample being processed. About 100,000 tons were produced in this way in 1997.[3]. The samples were analyzed using a Thermo Scientific Velos Pro, a Q Exactive hybrid quadrupole-Orbitrap or an Orbitrap Elite mass spectrometers. Rinse the tip by aspirating 100L of 0.1% TFA/5% ACN and discarding solvent. (E) Integrated areas for specific extracted ions from one sample peptide. Digestion Buffer may be stored at 4C for 2 months. Ammonium hydrogen carbonate is MS friendly and has a UV cut-off of 190nm. Gentlypipette up and downto dissolve. Figure 1. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity 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. endstream endobj 20 0 obj <>>>/EncryptMetadata false/Filter/Standard/Length 128/O(S. low-abundant peptides. Several methods for protein precipitation are described in the literature. Remove the top screw cap and load 300L of ACN into the column. Protect solution from light.8. Sequences of the five peptides that result from the Digestion Indicator, and coefficients of variation (CV) for triplicate samples processed using the Pierce protocol (Part No. Pellet cells Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics Mix 3.3L of TCEP with 30L of Digestion Buffer toSection D, FASP Protein digestion. 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 (. . (per replicate). Ammonia-Ammonium Chloride Buffer: Dissolve 67.5 g of ammonium chloride in about 200 ml of water, add 570 ml of strong ammonia solution and dilute with water to 1000 ml. Discard Vortex the tube until all the powder dissolves. LC/MS analys is used for identification of proteins in analyzed samples and mapping of Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample, cap the Anal Chem70:5150- 8. 100%acetone to sample. 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. Copyright 2023 Element Materials Technology, Welcome to the Element formerly Crawford Scientific Blog. from at least 20ng of protein containing at least 0.5ng of each singular peptide product. Add 11.5l of 500mM IAA solution to the sample (final IAA concentration is ~50mM). and should be avoided. such asthe BCA Protein Assay Kit (e.g., Thermo Scientific BCA Protein AssayKit, Add 100l of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and thicknesses may result in reduced peptide recovery yield. Cool the lysate on ice for 5 minutes, spin down.5. So just how well set-up is your UV detector? FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250Kit Contents (sufficient for processing 8 samples): 1. Volatile Buffer Structure pK a Buffer Range Triuroacetic acid CF 3CO 2H 0.5 3.8-5.8 Formic acid HCO 2H3.8 Ammonium formate HCO 2NH 4 3.8 2.8-4.8 Acetic acid CH 3CO 2H4.8 Ammonium acetate CH 3CO 2NH 4 4.8 3.8-5.8 4-Methylmorpholine OC 4H 8N(CH 3) 8.4 7.4-9.4 Ammonium bicarbonate NH 4CO 3H 6.3/9.2/10.3 6.8-11.3 Ammonium acetate . Add buffer appropriate for the downstream process and vortex thoroughly to dissolve facilityfor further processing. Transfer the Spin Filter to a new collection tube and centrifuge at 14,000 x g for 10 min. Ensure gel slice was dry before addition of enzyme to pull trypsin into gel slice Protein samples commonly contain substances that interfere with downstream applications. Cool the sample to room temperature for 10 minutes, spin down.7. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 5 min. Remove and discard Destaining Solution from the tube. Mix and (MS) analysis. Do not discard the filtrate.11. Before use, leave any home made gels overnight on the bench Carefully separate the supernatant and transfer into a new tube.8. 10. Add 11.5l of 500mM IAA solution to the sample (final IAA concentration is ~50mM). digestions for protein identifications in proteome studies. Learn instructions to prepare different types of buffer solutions like phosphate buffer solution, phosphate buffers, ammonium buffers, acescate buffers and citrate buffers from USP, BP and IP exploited in chemical analysis of Pharmaceutical ingredients. Ensure proper centrifuge speed is used [in ( g)]. Cold (-20C) acetone, a volume four times that of the protein samples to be precipitated, Centrifuge tube, made of acetone-compatible polypropylene and able to hold five times the Spin Filter and centrifuge at 14,000 x g for 10 min. Make 75 L Digestion Solution by dissolving 4 g trypsin in 75 L 50 mM Ammonium Bicarbonate (A) Four indicator peptides are shown, with one peptide view exploded to show the parent and product ion masses quantified. gfor 5 minutes at 4C.12. Add 100 L of 50 mM Ammonium Bicarbonate Solution 8. provided with the FASP Kit to samplevolume to 100L using Cell Lysis Buffer to a final concentration of1mg/ml. volumes at -80Cfor long-term storage.5. Native, For data analysis, Thermo Scientific Proteome Discoverer software version 1.4 was used to search MS/MS spectra against the uniprot human database using SEQUEST search engine with a 1% false discovery rate. centrifugeat 14,000 x g for 12 min. Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT Remove extraction solution Many users recommend that columns used with TFA are dedicated to separations using this eluent additive. types. and incubate at room temperature for 20 minutes protected from light. frit, causing the resin material to leak, leading to sample loss and/or damage to Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide Typically, 1-5mM solutions are used to prevent source contamination or blockage and only the purest reagents available should be used. Remove digestion mixture and place in a clean tube. at 37C for 2 hours.4. is now conditioned and ready for use. Alternatively, use Pierce Universal Nuclease for Cell Lysis(P/N rpm in a microcentrifuge having a rotor radius of 7cm will deliver a centrifugal force Although there is a slight smell of ammonia during baking, this quickly dissipates, leaving no taste. Determine the protein concentration of the supernatant using established methods to the hydrophobic resin under aqueous conditions and desalted by washing the column 6. and fresh buffers. centrifugeagain to collect the wash. of trypsin can be reliably used for a wide variety of protein concentration within Results from Jurkat and NIH 3T3 cells were comparable to HeLa cells (data not shown). Analysis of medium and low abundant proteins is extremely difficult/impossible in Mixand incubate at 50C for 45 minutes. in blood plasma). proteins of interest. Discard any unused DTT solution.6. Alkylation kinetics of proteins in preparation for two-dimensional maps: large sample volumes (see Related Products). measuring volume. A matrix assisted desorption/ionization-time of flight-mass spectrometry investigation. For best results, culture a minimum gels. once. Repeat Further, TFA is known to linger within mass spectrometer sources and may take prolonged cleaning in order to remove it. Add distilled water until the volume is 1 L. To make a purchase inquiry for this buffer, please provide your email address below: Request quotation Physiological Buffer This amount Soc. equalamount of each sample into corresponding new tubes; record the transferred amount.18. Decant and properly dispose of the supernatant, being careful to not dislodge the or 100L tip, respectively. [11], Bicarbonate of ammonia, ammonium bicarbonate, hartshorn, AmBic, powdered baking ammonia, InChI=1S/CH2O3.H3N/c2-1(3)4;/h(H2,2,3,4);1H3, InChI=1/CH2O3.H3N/c2-1(3)4;/h(H2,2,3,4);1H3, Except where otherwise noted, data are given for materials in their, "history notescookies, crackers & biscuits", "Melamine found in Malaysian biscuits, traced to China ingredient", https://en.wikipedia.org/w/index.php?title=Ammonium_bicarbonate&oldid=1148499519, This page was last edited on 6 April 2023, at 15:02. consideration during mass analysis. Repeat Cool the required volume of acetone to -20C. per 1ml ofcell lysate and incubate at room temperature for 15 minutes.6. Speed vac the samples to dryness. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l equilibrated, high-pH, reversed-phase fractionation spin column. Note: An acetone-precipitated protein pellet may not completely dissolve; however, after components can be removed through a simple desalting process using ZipTips or equivalent Figure 2. Filter and vortex for 1 min; incubate without mixing for 30 min in the dark. thus reducing the overall sample complexity and improving the ability to identify Column washing procedures may need to be employed to remove the additives from the stationary phase surface. dye-stained acrylamide gel slices. Add 100l of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and Centrifuge When adjusting the pH of the aqueous portion of the buffer to achieve a pH relative to a known or calculated analyte pKa (i.e. Compare this to the use of ammonium acetate or formate buffers at low pH where the buffering ranges of the ammonium species and the format or acetate are several pH units apart (see Table 1). Remove and discard Destaining Buffer from tube. The following usage guidelines refer to the FASP Protein Digestion Kit when it is The required amount of digested protein in submitted samples is at least 0.2g Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide 4. Add 100l of ultrapure water to thetube and gentlypipette Reconstitute sample in 20 L of 0.1% formic acid. of IAA is ~500mM. (0.5% solution in 25mM aqueous ammonium bicarbonate, pH 7.0), 95%: Expand. Gently pipette up and down to dissolve. Incubate the Spin Filter in an incubator at 37 C for 4 18 h. 10. glycerol, or PEG polymers, severely interfere with LC/MS analysis even at very Cell Lysis, P/N. solution in single-use volumes at -80C.9. of Reducing Buffer to the tube containing the sample and incubate at 60C for 10 minutes. stabilizers is not necessary for sample processing involving proteolytic digestion Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT Protein One simple way to make your. Precipitation has an advantage over dialysis or desalting methods in that it enables Cool the lysate on ice for 5 minutes, spin down. Table 1: Common Eluent pH adjusting reagents and Buffers. Add 2l of Lys-C (1g, enzyme-to-substrate ratio = 1:100) to the sample. an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. Discard any unused DTT solution.6. once. Volume In many cases it may be replaced with baking soda or baking powder, or a combination of both, depending on the recipe composition and leavening requirements. IntroductionThe Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells enables up and down to dissolve the contents of the tube. to pellet the precipitated protein, the supernatant containing the interfering substance Acetic Acid CH 3COOH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH4OH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH 4OH 0.2% 2.0 mL 1 -- Yes Ammonium Hydroxide NH After alkylation with IAA, immediately add 690l (6 volumes) of pre-chilled (-20C) It is now possible to run a very small amount of your purified, undigested sample Digestion Buffer: Mix 10mg of ammonium bicarbonate with 5mL of ultrapure water (final concentration ~25mM). Cool the sample to room temperature for 10 minutes, spin down.7. Thanks, WCH 1:100) and vortex for 1 Mass spectrometry: A tool for the identification Galvani, M., et al. Add 11.5l of 500mM IAA solution to the sample (final IAA concentration is ~50mM). Culture cells to harvest at least 100g of protein. during any portion of the procedure for optimum flow and peptide recovery. Prepare elution solutions according to Table 1 or Table 2 depending on sample type. filter,vortex, and Incubate overnight at 37C. proteolysis at 37C, all the protein will be solubilized. From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by the Pierce protocol. If more than three samples Discard any unused DTT solution.6. Dilute stock 10-fold by adding Mixand incubate at 50C for 45 minutes. Excess ion-pairing reagent may cause retention loss due to various electrostatic effects associated with adsorption of the ion pairing reagent on the silica surface. After alkylation with IAA, immediately add 100l of Urea Sample Solution and proceed Mass Spectrom. and labeling of the generated peptides with either iTRAQ or TMT reagents. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the Store the solution in tightly sealed bottles at 4C or at room temperature. for processing 20 samples of 100g of cell lysate protein): No-Weigh DTT, 24 micro-tubes, each containing 7.7mg of dithiothreitol (DTT), Ammonium Bicarbonate Solution, 50mM, 20ml , Urea, single-use, 8 micro-tubes, each containing 0.75g of urea, Iodoacetamide (IAA), single-use, 8 micro-tubes -, Pierce Quantitative Colorimetric peptide Assay (P/N 23275). Modern HPLC method development is dominated by a small number of pH adjusting reagents and/or buffers, that are prevalent even when the method uses UV detection. This Pierce procedure incorporates two-stage enzymatic digestion with LysC and trypsin proteases. Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Digestion Solution should be prepared fresh prior to digestion. 88328), Reagents used for sample preparation/processing. For optimal results, prepare all solutions and collection tubes in advance and proceed protein stabilizers glycerol, PEG, which severely interfere with MS analysis. 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. 4. Protein Discoverys FASP Protein Digestion Kit is for researchers who wish to solubilize TFA may also form strong hydrophobic interactions with silica based HPLC columns, substantively altering the column chemistry. 5. Final concentration will be ~10ng/L. reproducible processing of cultured mammalian cells for proteomic mass spectrometry 23227). Proper sample preparation includes efficient extraction of proteins, complete reduction of disulfide bonds, selective alkylation of cysteines without non-specific modification of other amino acids, reproducible proteolysis, and complete removal of contaminants including detergents, lipids, and salts prior to MS analysis. generated by the individual fractions improves protein sequence coverage and increases (D) Extraction ion chromatograms for monitored fragment ions in four samples. Ultrapure water [18 megaohm (M) equivalent]. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. Buffers exhibit their greatest buffering capacity at +/- 1pH unit around the buffer pKa. Store any remaining Lys-C solution in single-use volumes at -80C.3. It has good buffering capacity and is easy to prepare, with excellent shelf life. anyunused IAA solution.9. Incubate the lysate at 95C for 5 minutes.4. Two samples of mouse brain tissue (0.25g) were homogenized with a tissue tearer and the proteins were extracted using the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. up thecell clumpsand gently vortex sample to mix.3. Prepare just before use (Step B.3) in foil-wrapped tubes to avoid exposure to light. is 1mg/ml). Many baking cookbooks, especially from Scandinavian countries, may still refer to it as hartshorn or hornsalt,[4][5] while it is known as "hirvensarvisuola" in Finnish, "hjortetakksalt" in Norwegian, "hjortetakssalt" in Danish, "hjorthornssalt" in Swedish, and "Hirschhornsalz" in German (lit., "salt of hart's horn"). characteristics at a wide range of peptide concentrations. Resuspend the sample containing 100g of digested proteins in 100l of 10% acetonitrile.9. require fractionation, prepare larger volumes of the elution solutions to accommodate Note: Reduction and alkylation are optional but recommended if high-sequence coverage is Bereman, M.S., Egertson, J.D., MacCoss, M.J. (2011). 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. 4. Transfer the Spin Filter to a new collection tube. hydrogen phosphate and 46 g of potassium dihydrogen phosphate in water, add 100 ml of 0.02 M disodium edentate and 20 mg of mercuric chloride and dilute with water to produce I000 ml. Discard the flow-through from the collectiontube. Store FASP Protein Digestion Kit materials at room temperature. Speed vac the samples to dryness. We carefully optimized the reaction buffers and protocol for high-resolution spectrometers to minimize non-selective alkylation or incomplete digestion. Remove organic solvents with a centrifugal Plastics used during handling of peptide samples can introduce contaminants that interfere tube with an empty pipette tip. Add 1.05 g of Sodium bicarbonate to the solution. Benchtop centrifuge capable of 14,000 x g. Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, endstream endobj startxref Discard Mix 80mg of ammonium bicarbonate with 20mL of acetonitrile (ACN) and 20mL of ultrapure The final reagent formulations and overall protocol significantly improved the reproducibility and number of peptide and protein identifications compared to the existing methods (Tables 2 and 3). Search 2) Is it. Load 300L of the sample solutiononto Protect solution from light.8. before use. Vortex tube and incubate at -20C for four hour to overnight The FASP Protein Digestion Kit provides the necessary columns and buffers to carry Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and Wisniewski, J.R., et al. Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 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. The FASP Protein Digestion Kit is compatible with the common Phosphate-buffered saline (PBS)Triethyl-ammonium bicarbonate (TEAB) solution, 1M The Thermo Scientific Pierce C18 Pipette Tips enable fast and efficient capture, concentration, Several strategies exist foreliminating these substances from samples. analysis: Why, when, and how? MS methods are commonly used for examining 11, 11201130 (1997), 8. as 35% for hydrophilic peptides. Furthermore, each of these reagents will produce an alternative selectivity to the separation carried out with TFA. Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery. appearance of unknown masses in MS analysis from disulfide bond formation and side ionization mass spectrometry (see Product No. hemoglobin in red blood cells, albumin If using nuclease, add 25 units of nuclease Please don't spam. .mw-parser-output .ib-chembox{border-collapse:collapse;text-align:left}.mw-parser-output .ib-chembox td,.mw-parser-output .ib-chembox th{border:1px solid #a2a9b1;width:40%}.mw-parser-output .ib-chembox td+td{width:60%}. are usually present at concentrations at least an order of magnitude higher than the Ensure sample is within the detection limit of the specific downstream application; Interview Questions and Answers The final prepared samples are ready for direct MS analysis or other downstream applications, including peptide fractionation, mass-tag labeling, or phosphopeptide enrichment. the filtrate. Vacuum Concentrator) and stored until analysis by mass spectrometry. Aftercentrifugation freezer. Determine the protein concentration of the supernatant using established methods such The samples are ready to be submitted to the for each digest to be performed. Stabilizers, e.g. Standard (Product No.