phosphorylated, Thermo Scientific Tandem Mass Tag (TMT)-labeled, and other complex post-translational modifications (PTM) in identified proteins. (e.g., 2-D electrophoresis sample buffer, SDS-PAGE sample buffer, Pierce. step before LC-MS analysis. Hela lysate samples (10g-5mg) were prepared according to the Pierce protocol (Part No. centrifugeagain to collect the wash. Typically, 1-5mM solutions are used to prevent source contamination or blockage and only the purest reagents available should be used. per condition. All samples to be processed in the PMC MUST be run on the Agilent Bioanalyzer in the toSection D, FASP Protein digestion. Excess iodoacetamide and other contaminants were removed by acetone precipitation at -20C for 1 hour. and clean-up for peptide sequencing. Note: An excess of Digestion Buffer is supplied to minimize the need for long-term storage is 1mg/ml). Then, 100g of lysate was processed according to the kit procedure, and 500ng samples were analyzed by LC-MS/MS on a Thermo Scientific Velos Pro ion trap mass spectrometer. (mBIO) core (x8-3743) if you are unsure about statistical requirements for an experiment. Remove and discard Destaining Buffer from tube. The complete Pierce Mass Spec Sample Prep Kit for Cultured Cells includes Lysis Buffer, Digestion Indicator, Reaction Buffers, Proteases and with instructions to process up to 20 samples. Mixand incubate at 50C for 45 minutes. Wisniewski, J.R., et al. amount of reagents (DTT, IAA, Lys-C and trypsin). Note: The recommended amount of trypsin used per digest is 100ng (see protocol). 1. 88700) toenzymatically digest DNA and RNA. DMSO, DMF interfere with MS analysis. The column Ammonium bicarbonate is an irritant to the skin, eyes and respiratory system. Replace the cap, place activity that should not interfere with mass spectral analysis. An automated multidimensional protein identification technology for shotgun proteomics. unusedIAA solution.9. Note: An acetone-precipitated protein pellet may not completely dissolve; however, after Speed vac the sample (106l) for at least 2 hr. We carefully optimized the reaction buffers and protocol for high-resolution spectrometers to minimize non-selective alkylation or incomplete digestion. Native, Add 200l of Urea Sample Solution to a Spin Filter and centrifuge at 14,000 x g for Sechl, S. and Chalt, B. T. (1998). 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. Peptide fragments with one missed cut are common and should be taken into Store buffers at 4C. The kit contains all of the necessary buffers, reagents, MS-grade enzymes; Add buffer appropriate for the downstream process and vortex thoroughly to dissolve Add 100 L of 50 mM Ammonium Bicarbonate Solution 9. provided with the FASP Kit to We recommend the preparation for just 4 . To avoid inaccurate volumetric dispensing, do not use Pierce C18 Pipette Tips for Sample should look cloudy. 5. Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the 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. Discard any unused DTT solution.6. buffers in glass vessels. Speed vac the sample (106l) for at least 2 hr. (2001). Gentlypipette up and downto dissolve. in blood plasma); selective depletion of abundant proteins (to at least average abundance Culture cells to harvest at least 100g of protein. Decant and properly dispose of the supernatant, being careful to not dislodge the Thermo Fisher Scientific, byBabu Antharavally, Ph.D.; Xiaoyue Jiang, Ph.D.1; Robert Cunningham, Ph.D.; Ryan Bomgarden, Ph.D.; Yi Zhang, Ph.D.1; Rosa Viner, Ph.D.1; John C. Rogers, Ph.D.- 06/04/13. Discard any unused DTT solution.6. Ready to use SOPs, Protocols, Master Plans, Manuals and more Worldwide Regulatory Updates Detergents Note: This procedure is optimized for 100g of cell lysate protein at 1mg/mL concentration; Discard the flow-through from the collection tube. acetone with 5mL of ultrapure water) and store at -20C. 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. centrifugeat 14,000 x g for 10 min. Do not introduce air through the membrane x. 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. of CellLysis Buffer for a 20l cell pellet). Usually, use of protein Another strategy for removing undesirable A similar decomposition takes place when the sesquicarbonate is exposed to air. The compound has many names, reflecting its long history. that inhibit trypsin digestion, b) compounds with primary Ammonium bicarbonate decomposes above about 36 C into ammonia, carbon dioxide, and water in an endothermic process and so causes a drop in the temperature of the water: When treated with acids, ammonium salts are also produced: It reacts with sulfates of alkaline-earth metals precipitating their carbonates: It also reacts with alkali metal halides, giving alkali metal bicarbonate and ammonium halide: The compound occurs in nature as an exceedingly rare mineral teschemacherite. endstream endobj 20 0 obj <>>>/EncryptMetadata false/Filter/Standard/Length 128/O(S. to remove the (volatile) Digestion 3 . Centrifuge the Spin Filter at The maximum loading capacity of one FASP Protein Digestion Kit is 0.4 mg protein in Cool the sample to room temperature for 10 minutes, spin down.7. analyze the resulting peptides by mass spectrometry. It was obtained by the dry distillation of nitrogenous organic matter such as hair, horn, leather. ionization mass spectrometry (see Product No. 8. Centrifuge lysate at 16,000 g for 10 minutes at 4C.7. The required amount of digested protein in submitted samples is at least 0.2g 5. Analysis of medium and low abundant proteins is extremely difficult/impossible in For example, centrifugation of a sample at 5,000 Final concentration will be ~10ng/L. Store any remaining trypsin ZipTip columns are available for resale in the PMC. Carefully remove acetone withoutdislodging Mixand incubate at 50C for 45 minutes. column into a 2.0mL sample tube. to LC/MS analysys. Synonym(s): Hartshorn salt . 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. This stock solution can be prepared three times with this kit. rpm in a microcentrifuge having a rotor radius of 7cm will deliver a centrifugal force Wet tip by aspirating 10L of 50% ACN in water and then discarding solvent. The methodology only the number of cycles necessary for the application. Load 300L of the appropriate elution If more than three samples P/N 23227). glycerol, or PEG polymers, severely interfere with LC/MS analysis even at very Solubilize the pellet in buffer appropriate for downstream process. Add 100 L of Urea Sample Solution to the Spin Filter and 7. centrifuge at 14,000 3. This solution is used to form the Trypsin Working Solution as needed (see It is used in, for example, Swedish "drmmar" biscuits and Danish "brunkager" Christmas biscuits, and German Lebkuchen. FASP columns) or by acetone precipitation. Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Mix and dissolve the solution by pipetting for each digest to be performed. such asthe BCA Protein Assay Kit (e.g., Thermo Scientific BCA Protein AssayKit, Features of the Mass Spec Sample Prep Kit: Learn more about the Thermo Scientific Pierce Mass Spec Sample Prep Kit. [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. Repeat Editable Pharmaceutical Documents in MS-Word Format. be used at sufficient, but minimal, concentrations. S is the centrifuge speed in rpm. ==V2a>ls y9N`k@C* tb]L ;;s23;eoA0zF|:+-(zxxG,5z{q FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250Kit Contents (sufficient for processing 8 samples): 1. Speed vac the desalted sample to dryness.15. concentrations in a volatile high-pH elution solution is then applied to the columns Store any remaining trypsin Immediately before use, add 40L of ultrapure water to the bottom of the vial containing20g Lys-C and incubate at room temperature for 5 minutes. Try to stain gels for the minimum time needed until the band(s) of interest are visible, De-stain bands sufficiently to clearly see the band of interest edges, Save a high-quality image of the gel prior to band excision (optional), NEVER place gels on plastic for cutting , only cut gels on a clean glass plate, Gel band pieces are now ready for in-gel digestion and can be stored for 2-3 days H. 2. Detergents (both ionic and non-ionic) severely interfere with both LC and MS parts of analysis; the downstream application. pipette up and down to dissolve the contents of the tube. Oh well, back to ammonium bicarbonate. Retention time under reversed phase conditions will tend to increase with increasing ion pair chain length; however, care is required to add just enough ion pairing reagent for improved retention. 2. PierceDigestion Indicator per g of sample protein). P/N 23227), 5. Preparation of elution solutions for unlabeled, native peptides. The samples are ready to be submitted to the facility for LC/MS analysis. This solution may be stored at -20C for 2 months without Swell These components (except for enzymes) side of lysine and arginine residues. gfor 5 minutes at 4C.12. at14,000 x g for 15 min. The Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit provides components can be removed through a simple desalting process using ZipTips or equivalent In-gel digestion coupled with mass spectrometric analysis is a powerful tool for the Five digestion indicator peptides were quantified manually with extracted ion-chromatograms of the raw LC-MS/MS data or automatically with Thermo Scientific Pinpoint 1.2 software. The ammonium bicarbonate buffer also provides moisture during enzymatic cleavage. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N There is no absolute single best way to lyse cells and extract proteins. Approx. For the best experience on our site, be sure to turn on Javascript in your browser. Resuspend the sample containing 100g of digested proteins in 100l of 10% acetonitrile.9. A variety and Langen, H. (2000). Further, TFA is known to linger within mass spectrometer sources and may take prolonged cleaning in order to remove it. Therefore, use gels with 1 mm spacers (gel thickness) Phosphate-buffered saline (PBS)Triethyl-ammonium bicarbonate (TEAB) solution, 1M Iodoacetamide Solution by adding 100 L Urea Sample Solution to one tube of Iodoacetamide . The coefficients of variation (CV) for replicates of the five peptides were 5-16% with an overall mean CV of 10% (Table 4). Small soluble Use this 1M ammonium bicarbonate (20X) stock . It is insoluble in acetone and alcohols. This amount and incubate at 50C for 45 minutes. Add distilled water until the volume is 1 L. protocol for best results. .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%}. a proximal acidic, aromatic or proline residue; proline having the most significant Kit toone tube of Urea, also provided with the FASP Kit. (Optional) To further extract peptides, add 10L of 1% trifluoroacetic acid or 1% 7. 0.005g of Mix Pre-chilled 100% acetone: Store 100% acetone at -20C. Spin Filter and centrifuge Methanesulphonic acid (MSA) can also be used as a very effective alternative to TFA when using UV detection. up thecell clumpsand gently vortex sample to mix.3. Chem. Autosampler vials or 0.5mL, 1.5mL microcentrifuge tubes, Wetting solution: 50:50 ACN:water; 20L or 200L per sample, Equilibration solution: 0.1% TFA in water; 20L or 200L per sample, Rinse solution: 0.1% TFA in 5% ACN:water; 20L or 200L per sample, Elution solution: 0.1% TFA in 50-95% ACN:water for MALDI-MS or 0.1% FA in 50-75% ACN:water vacuum evaporator but avoid complete dryness, which might result in sample loss. Retain eluate as wash fraction. protein extracts are then dissolved and trypsin digested in an appropriate buffer. Solution provided with the FASP Kit. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.7. The protein was resuspended in digestion buffer and digested with Lys-C (1:100, enzyme:substrate) for 2 hours at 37C followed by digestion with trypsin (1:50, enzyme:substrate) overnight at 37C. For example, to test reproducibility of our optimized method, we processed and analyzed quadruplicate samples of a HeLa cell culture using the Pierce protocol, spiking the Digestion Indicator into each lysate after the initial lysis step (same method as for Table 3). Rapid Commun. for 5 minutes. 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 However, this has been shown not to be the case in many examples and has come to be known as wrong way round ionisation [7,8]. Carefully remove acetone without dislodging the protein pellet. primarily for MS applications, they may be used for applications such as peptide concentration Figure 5. Ensure sample is within the detection limit of the specific downstream application; proteins of interest. To develop the Pierce protocol, we first used a step-wise approach to optimize a cell lysis method to maximize protein extraction and recovery from the resulting lysate. Electrophoresis22:2058-65. Interview Questions and Answers Compare Product No. A variety of Thermo Scientific dialysis and Product Usage Information. agents, detergents, etc. appearance of unknown masses in MS analysis from disulfide bond formation and side FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250Kit Contents (sufficient for processing 8 samples): 2. Determine the peptide concentration in the samples using Pierce QuantitativeColorimetric One further note on MS signal intensity is the use of forced adduct formation to improve the sensitivity of the analyte, or to distinguish one analyte from another within the MS chromatogram. After alkylation with IAA, immediately add 100l of Urea Sample Solution and proceed Dilute stock 10-fold by adding The final concentration of DTT is~500mM. Add 40 L of 50 mM Ammonium Bicarbonate Solution to the 13. the Spin Filter at 14,000 x. Filtrate contains digested proteins. to elute bound peptides into eight different fractions collected by centrifugation. for optimum tip-to-pipettor seal and sample aspiration. sorbent that minimizes flow resistance and provides excellent binding and recovery Add four times the sample volume of cold (-20C) acetone to the tube. centrifugeat 14,000 x g for 10 min. Proteomics2:1630-2. So just how well set-up is your UV detector? It has been assigned E number E503 for use as a food additive in the European Union. The standard pH values given in the tables and elsewhere in the Appendix are considered to be reproducible within 0.02 unit at 25. 84840) Samples were incubated at 95C for 5 minutes except the urea sample, which was incubated at RT for 30 minutes. Incubate the lysate at 95C for 5 minutes. Therefore, at pH 8.5, you will have both carbonate and bicarbonate present. Digestion indicator peptides were quantified with Thermo Scientific Pinpoint 1.2 software, which is pre-programmed with information on the Digestion Indicator peptides and MS2 transitions to quantify (Figure 3). Ammonia gas passed into a strong aqueous solution of the sesquicarbonate (a 2:1:1 mixture of (NH4)HCO3, (NH4)2CO3, and H2O) converts it into normal ammonium carbonate ((NH4)2CO3), which can be obtained in the crystalline condition from a solution prepared at about 30 C. Vortex tube and incubate at -20C for four hour to overnight 2. Required components Prepare 800 mL of distilled water in a suitable container. A step gradient of increasing acetonitrile Mass spectrometry: A tool for the identification 45 0 obj <>stream 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. Add 100g of lysate protein to a polypropylene microcentrifuge tube and adjust the However, because some sample loss will accompany each cycle ofprecipitation, use Ammonium hydrogen carbonate has been described as an excellent buffer for the analysis of basic drugs by HPLC-MS (10). This is especially useful in the analysis of peptides and proteins and typically 5mM of medronic acid can be added to buffered mobile phase (ammonium acetate for example) to provide highly-effective deactivation, resulting in improved peak shapes, detector sensitivity, and quantitative reproducibility. Pharmaguideline is a pharmaceutical blog where pharmaceutical concepts are explained in very simple and easily understandable language for professionals and students. analysis. as well. measuring volume. Hodges, Journal of Chromatography A, 1080 (2005) 6875, 5. Using the buffer preparation calculator. Speed vac the sample (206l, containing ~ 100g of digested proteins) to ~20-50l Place the column into a new 2.0mL sample tube. It has good buffering capacity and is easy to prepare, with excellent shelf life. Aebersold, R., and Mann, M. (2003). at any time during the procedure. Store buffers at 4C. Screenshot of software analysis for indicator peptides. as 35% for hydrophilic peptides. Match Criteria: Product Name, Keyword. This quantitative analysis further demonstrated the high reproducibility of sample processing using the optimized protocol. The 100L tip allows processing of CellLysis Buffer for a 20l cell pellet). BioAssays. 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. 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. A tool in peptide mapping and protein identification. Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery. processing, Sample not sufficiently hydrophobic to bind C18 sorbent, Peptides binding to plastics can cause significant loss at low peptide concentrations, Minimize contact with plastics, excessive drying and storage at low concentrations Standard Buffer Solutions are solutions of standard pH. This analysis indicated <10% missed cleavages. Detergents can be successfully removed before proteolytic digestion of proteins using tominimize the effects from evaporation.10. Learn how to prepare different types of buffer solutions like phosphate buffer solution, ammonia buffers, ammonium buffers, acetate buffers and citrate buffers from USP, BP and IP used in chemical analysis of Pharmaceutical ingredients. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100l the powder dissolves. 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. the pellet difficult to re-solubilize.Therefore, use precipitation only for downstream The following usage guidelines refer to the FASP Protein Digestion Kit when it is Transfer the Spin Filter to a new collection tube. large sample volumes (see Related Products). Vortex tube and incubate at -20C for four hour to overnight Peptide samples were also prepared according to standard urea, FASP1, and AmBic/SDS2 methods. Diagram of the developed protocol. Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of100% These reagents also linger for much shorter times within ESI sources. the filtrate. Discard the flow-through from the collection tube. (E) Integrated areas for specific extracted ions from one sample peptide. the manufacturers protocol.14. The extended buffering range is due to the ammonia - ammonium buffering capacity being additive to the hydrogen carbonate-carbonate capacity in what is traditionally called a 'mixed . Protein extracts can be separated from these low MW components by filtration using Protect solution from light.8. Each of these common protocols has disadvantages; FASP requires many long centrifugation steps, SDS-based methods may not be scalable and require detergent removal from peptides, and urea must be made fresh and can carbamylate lysine residues. All articles and SOPs are written by Ankur Choudhary. 4. low concentrations and are difficult to remove from prepared samples. 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. Load 300L ofwater onto the columnand Preparation of Bicarbonate-Carbonate Buffer Solutions (pH 9.12-10.83) Solution A: 0.1M sodium bicarbonate (NaHCO3 MW = 84.0) (8.40 g/L) References:. This protocol also includes a unique, non-mammalian internal digestion control standard protein (Digestion Indicator) to assure protocol performance and to quantify sample preparation processing and digestion efficiency across samples. Add 200L of Destaining Solution to gel pieces. The samples are ready to be submitted to the For optimal results, prepare all solutions and collection tubes in advance and proceed Final TCEP concentration is ~50mM. Mansoori, B.A., Volmer, D.A., Boyd, R.K.: Wrong-way-round electrospray ionization of amino acids. Immediately before use, puncture the foil covering of the Thermo Scientific No-WeighDTT (C) Integrated area of the DGGYYSSVVDSHMHFK peptide transitions from four replicate samples. (0.5% solution in 25mM aqueous ammonium bicarbonate, pH 7.0), 95%: Expand. 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 pipette upand down to dissolve. in the Spin Filter and centrifuge at 14,000 x, Add 200 L of Urea Sample Solution to the Spin Filter and 2. centrifuge at 14,000 Add 50l of pre-chilled (-20C) 90% acetone, vortex to mix and centrifuge at 16,000 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]. dimensions: 1mm X 1mm X 5mm. in blood plasma). 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 . Carefully separate the supernatant and transfer into a new tube. solvents such as acetonitrile (ACN) or methanol. Cool the lysate on ice for 5 minutes, spin down.5. The FASP Protein Digestion Kit is compatible with the common of Alkylation Buffer to the tube. once. Trypsin into four separate tubes of ~5L each. As a very approximate rule of thumb follow these guidelines; Remember that trifluoroacetic acid (TFA) is a strong ion pairing reagent and may severely restrict the detector sensitivity in positive ion mode, because the ion pair is strong enough to survive as a neutral complex with the analyte during liberation into the gas phase. Protein If local exhaust ventilation or enclosure is not used, respirators are necessary. It may be used to coat 2D and 3D electrophoretic gel pieces during digestion of proteins and proteomes. protein pellet. the presence of highly abundant proteins (e.g. lysates from a wide variety of biological sample types. Prepare 10mL of equilibration solution by adding 10L of TFA to 10mL of water. Copyright 2023 Element Materials Technology, Welcome to the Element formerly Crawford Scientific Blog. Cell Lysis, P/N.

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