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Sample Cleanup

Sample Cleanup. Voyager Training Class. Compound. Concentration. Peptides and proteins. 0.1 to 10 pmol/µL. Oligonucleotides. 10 to 100 pmol/µL. Polymers. 100 pmol/µL. Sample Dilution/Concentration

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Sample Cleanup

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  1. Sample Cleanup Voyager Training Class

  2. Compound Concentration Peptides and proteins 0.1 to 10 pmol/µL Oligonucleotides 10 to 100 pmol/µL Polymers 100 pmol/µL Sample Dilution/Concentration Dilute samples to the concentrations shown in the table below. If the sample concentration is unknown a dilution series may be needed to produce a good spot on the MALDI plate. Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.

  3. Appearance of Matrix/Sample Spot Contamination Effects • Ring effect around the crystallized matrix • Clumping of matrix in the well • Matrix does not crystallize • Droplet spreads over wide area • from matrix, sample or solvent(s)

  4. Sample clean-up • Removal of buffer salts, urea, guanidine, EDTA, glycerol, DMSO, detergents, etc. • Dilution • Washing • Drop dialysis • Cation exchange • Pipette tip column chromatography • ZipTips

  5. Sample Dilution Simplest way to minimize effect by contaminants. Goal is to dilute contaminants to the point where they no longer interfere with analysis of sample. Requires high enough analyte concentration in sample to provide acceptable data when diluted out.

  6. Typical contaminants in protein/peptide samples No interference: TFA, formic acid, b-mercaptoethanol, DTT, volatile organic solvents, HCl, NH4OH, acetic acid Tolerable:(< 50 mM) HEPES, MOPS, Tris, NH4OAc, octyl glucoside Note: Minimizing buffer concentrations improves performance. Use the minimum needed to control pH. Avoid: glycerol, sodium azide, DMSO, SDS, phosphate, NaCl, 2M urea, 2M guanidine Reference: Swiderek, K, Alpert, A, Heckendorf, A, Nugent, K, Patterson, S; Structural Analysis of Proteins and Peptides in the Presence of Detergents: Tricks of the Trade; ABRF News, Methods and Reviews, Dec. 1997,17-25

  7. On-Plate Washing Buffer and Salt Removal • Dry sample and matrix • Deposit 1-2 uL cold 0.1% TFA • Leave on for 5-10 sec., then remove Detergent contamination • Use 5% Isopropanol Cell Extract Contamination • Use 100% Isopropanol

  8. Drop Dialysis To remove low molecular weight contaminants Use Millipore membrane, type VS, pore size 0.025 mM, diam. 25 mm • Fill a 250-400 mL container with deionized water. • Float the membrane on the water (shiny side up). • Place about 10 uL of sample solution on the membrane. • Add 1uL ACN to the sample spot to increase surface area. • Allow to sit for ~45 minutes. • Remove an aliquot with pipette, add matrix and spot plate. H. Goerisch, Anal. Biochem.173, 393-398 (1988).

  9. After Before Drop dialysis cleanup of Enolase Yeast Enolase (47 kDa) in 8 M urea was dialyzed for 1 hr on a Millipore membrane.

  10. Cation Exchange BeadsFor removal of alkali metal ions Preparation of resin in the NH4+ form: • Use Dowex cation exchange resin 50WX8-200, 8% crosslinked, H+ loaded (can purchase from Sigma) • Stir resin beads in 2x volume of 1M NH4Acetate overnight. • Filter, wash with deionized water, acetone and hexane. • Dry and store for use.

  11. Cation Exchange Beads • To use resin: • Place ~0.1 mg of beads on a clean piece of Parafilm. • Add 5 uL of sample and an equal amount of matrix to the beads to make a slurry of approx. 50% beads. • Slowly mix up and down with the pipette 10-15 times. • Allow the beads to settle for 15-30 sec. • Pipette supernatant onto the sample plate. • Change tip to avoid carrying over beads to sample plate.` Note: Do not use with positively charged species!

  12. Cation exchange bead cleanup of 31-mer oligonucleotide Multiple Sodium Adducts No cleanup 1500 1000 Counts After cation exchange 500 8500 9000 9500 10000 10500 11000 11500 Mass (m/z) The 31-mer was originally in PBS/2M NaCl.

  13. Sample Cleanup by Solid Phase Extraction • ZipTip - miniature column chromatography 1. Standard ZipTip C18 have 0.6 ul bed volume 2. Micro ZipTip C18 have 0.2 ul bed, better for automation (less resistance to flow) 3. ZipTip C4 for cleanup of protein samples 4. Other types available, e.g. Metal Chelating (MC) for concentration of Phosphopeptides

  14. Micropipets Loaded with Packing Material: ZipTip C18

  15. Procedure for using ZipTip C18 Proteins/Peptides • Condition the ZipTip with 10 µl of acetonitrile (ACN), then 10 µl of 50% ACN/0.1% TFA, then 2 x 10 µl of 0.1% TFA. • Load the sample onto the ZipTip by pipetting 5-10 µl sample up and down several times and discarding the liquid. • Wash C18 tip with 3 x 10 µl of 0.1% TFA to remove salts. • Elute the sample from the ZipTip with 30-70% ACN or elute directly into the matrix (e.g. CHCA in 50% ACN/0.1%TFA); minimal volume of ~3 µl can be used.

  16. Use of the ZipTip C18 Maintenance of Sample Fidelity Initial feasibility studies were performed with simple protein and peptide mixtures. All peptides and proteins were retained by the tips with no significant difference between the standard preparation and the C18 ZipTip method

  17. Standard Preparation vs. ZipTip C18 for a Peptide Mixture ACTH 1-17 2.0 pmol/µl ACTH 18-39 1.5 pmol/µl ACTH 7-38 3.0 pmol/µl Angiotensin 2.0 pmol/µl Insulin3.5 pmol/µl 5000 Standard Prep 0 Counts -5000 ZipTip C18 Prep -10000 2000 3000 4000 5000 6000 Mass (m/z)

  18. Standard preparation vs. ZipTip C18 for a mixture of small proteins Apomyoglobin 4.0 pmol/µl Thioredoxin 2.75 pmol/µl Insulin 0.5 pmol/µl 20000 10000 Standard Prep 0 Counts -10000 ZipTip C18 Prep -20000 6000 8000 10000 12000 14000 16000 18000 Mass (m/z)

  19. Use of ZipTipC18 Sample Concentration and Buffer Removal Dilute samples can be concentrated by adsorbing analyte from multiple 10 l aliquots into the ZipTip and eluting out into a small volume, effecting a 10- to 50-fold concentration. Mild conditions (e.g. 0.1% TFA) will retain peptides and proteins on a ZipTip but remove common buffers and salts such as: 2M NaCl, 100mM Phosphate, 8M Urea, 6M Guanidine or 50% Glycerol

  20. 6000 ZipTip C18 Prep 4000 2000 0 Standard Prep Counts -2000 -4000 -6000 20000 30000 40000 50000 60000 70000 80000 90000 100000 Mass (m/z) Concentration and Buffer Removal of <0.01 mg/ml IgG HC by ZipTip C18 Preparation Analysis of IgG Heavy Chain in 0.2M Tris/ 6M urea at <0.01 mg/ml. A concentration effect was seen as a smaller volume of eluent was used than was drawn up.

  21. ZipTip C18 Prep in PBS/Urea/NaCl 40000 20000 0 Counts Standard Prep in PBS/Urea/NaCl -20000 -40000 1000 1500 2000 2500 3000 3500 4000 4500 Mass (m/z) ZipTip C18 Efficiently Removes Protein HC Digestion Buffer Analysis of a peptide map of IgG HC digest containing phosphate, NaCl, urea and DTT at 0.1 mg/ml digested with endo Lys C.

  22. Standard Prep Multiple Sodium Adducts 1500 ZipTip C18 Prep 1000 Counts 500 Dialysis Prep 8500 9000 9500 10000 10500 11000 11500 Mass (m/z) ZipTip C18 verses Floating Membrane Dialysis for Salt Removal from a 31mer Oligonucleotide The 31mer in PBS/2M NaCl was dialyzed for 6 hours. This is compared to the use of the ZipTip C18. The protonated peak of the 31mer was acquired using either of the desalting techniques.

  23. High Mass Calibrant: Enolase Enolase 2 Enolase 1 Photoadduct ZipTip C18 Preparation Standard Preparation Mass (m/z) 44000 48000 ZipTip C18 cleanup of Yeast Enolase reveals two components - Enolase 1 and 2. The high resolution obtainable (~500) with this method makes it a good high mass calibrant.

  24. Use of the ZipTipC18 Fractionation As peptides and proteins have differing affinities for the C18, the ZipTips can be used to fractionate mixtures according to their hydrophobicities. Increasing the ACN conc. in a step gradient of 10% -50% typically elutes out progressively higher masses. By fractionating a peptide mass map this can also be beneficial for PSD analysis. Step-gradient elutions of complex mixtures from the C18 tip result in high quality spectra and increased detection limits by reducing sample suppression.

  25. Run gel; stain, scan Proteomics : Experimental Approach Proteome indicates the proteins expressed by the genome or tissue 1000 1500 2000 Mass (m/z) Extract peptides; mass analyze Highly acidic/basic, hydrophobic or membrane proteins from MDLC Excise spot, wash, digest Database search

  26. In-Gel Digest Fundamentals Success depends upon: • Avoiding contamination of samples • Digesting the protein efficiently • Maximizing recovery of peptides • Minimizing losses from handling

  27. In-Gel Digest Method Handling the Gel and Slices • Gloves and lab coats must be worn at all times to avoid keratin contamination. Work on a clean surface. • Use clean polypropylene microcentrifuge tubes, 500 or 1500 ul with snap caps. Test first to confirm OK (i.e., does not leach out polymers, mold release agents, plasticizers, etc.) Set aside a box for digest use only, handle only with gloves.   • Use only clean tools, containers and reagents for anything that will come in contact with the samples.  • Keep samples capped at all times unless being processed.

  28. In-Gel Digest Method Note: Silver Stained Gels Non-destructive (i.e., no glutaraldehyde) Silver-stained samples should be trimmed and treated to remove the silver prior to washing as follows: Prepare stock solutions of 30 mM Potassium Ferricyanide and 100 mM Sodium Thiosulfate. Store each at 4C for up to 3 months. Make the working destain solution immediately prior to use by mixing the two stock solutions above at a 1:1 ratio. Trim the gels to 1 mm3 or less and soak in 100 ul destain solution for 10 minutes. This step converts the silver to a water soluble form. The gels will clear. Carefully remove the destain solution and wash 3X in dH20 (400 ul, 15 min. each) Use gel loading tips to prevent accidental aspiration of gel pieces. This step washes away the soluble silver. Ref: Electrophoresis 1999, 20, 601-605

  29. In-Gel Digest Method Washing Destained Silver and Coomassie Gels Trim the gel slices as needed to 1 mm3 or smaller. Run a negative and positive control, as well as a reagent control (containing no gel slice). Transfer gels to 500 or 1500 ul capped microcentrifuge tubes Wash gels 3X in 50% ACN/25 mM NH4 Bicarbonate pH 8.0 (400 ul, 15 min. each time). This removes gel contaminants and brings buffer into the gel. Soak in 100% ACN for 5 min. to dehydrate the gels, they will turn opaque white. Remove the ACN. (Note: Be sure that the ACN used does not contain any acid, otherwise the pH will be incorrect. Dry gels in Speed-Vac for 20-30 min. This will shrink the gels. (Be sure that the inside of the Speed-Vac is clean and free of particulates. Do not allow anyone to use the Speed-Vac with ungloved hands during this step as sample tubes will be uncapped).

  30. In-Gel Digest Method Enymatic Digestion – Trypsin Promega Sequencing Grade Modified Trypsin 10-15 ug/ml in 25 mM NH4 Bicarbonate pH 8.0. Store at -70C in one-time-use aliquots. (100 ul each) Rehydrate the dried gels with approx. 10-15 ul cold Trypsin solution. The gels will swell and turn clear. Check after 30 min. for sufficient volume to completely wet entire gel. Add additional Trypsin if needed for large gel pieces. There is no need to overlay with additional buffer. Incubate tightly capped at 37C for 16-24 hours. Convection oven is preferable to heat block.

  31. In-Gel Digest Method Extraction of Peptides Soak the gel slice in 25-50 ul 50% ACN / 5% TFA for 30-60 min. with gentle agitation. Do not vortex. Transfer the supernatant to a second clean tube . Extract the gel again with another 25-50 ul aliquot of 50% ACN/ 5% TFA for 30-60 min. Combine the two extracts and Speed-Vac to complete dryness, about 1 hour. Note: dry at room temp or heat to no more than 30C. Drying can also be done in a lyophilizer.

  32. In-Gel Digest Method Reconstitution Reconstitute the dried sample by adding 3.0 ul of 50% ACN/0.1% TFA to the bottom of the tube and gently pipetting up and down 4-5X to dissolve the extracted peptides. Do not vortex. Mix 0.5 ul reconstituted extract with 0.5 ul fresh -cyano matrix on a MALDI plate. Spot Cal Mix 1 adjacent to sample for close external calibration. Dry remaining extracts in Speed-Vac and freeze.

  33. In-Gel Digest Method MALDI-TOF Analysis Acquire a good spectrum in reflector mode with a method optimized for high resolution in 800-3000 Da range. Calibrate with internal Trypsin peaks T7 (842.5099) and T4 (2211.1046) if present, otherwise use close external calibration. Alternatively, samples can be spiked with dilute Cal Mix 1or 2 (approx. 1:500 in the matrix) for internal calibration. Finally, samples can be internally re-calibrated with known peak masses from a good Protein Prospector MS-Fit hit. If spectrum is poor due to contaminants or low peptide concentration try cleanup and/or concentration of the remaining extract with ZipTip C18

  34. Variations of the In-Gel Digest Steps Staining Procedure Results have shown that Coomassie Blue should be used if the sensitivity is adequate as the recovery of peptides is better than with Silver Staining. Excising the Gel Spot Care should be taken to cut precisely around the stained area to prevent any unnecessary contamination. Digestion Step Ammonium Bicarbonate should be used for MALDI compatibility. Tris is a good alternative. Low concentration can be used, 10mM is sufficient.

  35. Internal Calibration Masses Porcine Trypsin Peaks 842.5100 1045.5642 2211.1046 2283.1807 2299.1756 2239.1359 2807.3000 Bovine Trypsin Peaks 805.4167 906.5050 1153.5741 2163.0570 2273.1600 2289.1649 2530.3339

  36. Internal Calibration Masses Other Useful Peaks Internal Calibrants spiked into sample, e.g. Cal Mix1 @ 1:500 or 1:1000 dilution Keratin peaks if low intensity Common Keratin Peaks (delete from peak table before submitting to database search) 897.4140 1184.5911 1383.6909 2312.1482 973.5318 1193.6166 1434.7705 2383.9524 1037.5267 1234.6796 1474.7494 2510.1323 1060.5639 1307.6782 1474.7858 2705.1617 1066.4992 1320.5834 1699.8251 2831.1947 1066.5169 1357.7188 1707.7727 3312.3087 1140.5649 1357.6963 1716.8517 1165.5853 1365.6399 1838.9149 1179.6010 1373.6549 1993.9772

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