Sample Dilution/ConcentrationDilute 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-Vacor Solid Phase Extraction..
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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)
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
16. Use of the ZipTip C18
17. Standard Preparation vs. ZipTip C18 for a Peptide Mixture
18. Standard preparation vs. ZipTip C18 for a mixture of small proteins
19. Use of ZipTip C18
20. Concentration and Buffer Removal of <0.01 mg/ml IgG HC by ZipTip C18 Preparation
21. ZipTip C18 Efficiently Removes Protein HC Digestion Buffer
22. ZipTip C18 verses Floating Membrane Dialysis for Salt Removal from a 31mer Oligonucleotide
23. High Mass Calibrant: Enolase
24. Use of the ZipTip C18 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.
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
35. Internal Calibration Masses
36. Internal Calibration Masses