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
Voyager Training Class
Peptides and proteins
0.1 to 10 pmol/µL
10 to 100 pmol/µL
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.
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.
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.
glycerol, sodium azide, DMSO, SDS, phosphate, NaCl, 2M urea,
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
Buffer and Salt Removal
Cell Extract Contamination
To remove low molecular weight contaminants
Use Millipore membrane, type VS, pore size 0.025 mM, diam. 25 mm
H. Goerisch, Anal. Biochem.173, 393-398 (1988).
Drop dialysis cleanup of Enolase
(47 kDa) in 8 M urea was dialyzed for 1 hr on a Millipore membrane.
Cation Exchange BeadsFor removal of alkali metal ions
Preparation of resin in the NH4+ form:
Note: Do not use with positively charged species!
Multiple Sodium Adducts
After cation exchange
The 31-mer was originally in PBS/2M NaCl.
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
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
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
ZipTip C18 Prep
Apomyoglobin 4.0 pmol/µl
Thioredoxin 2.75 pmol/µl
Insulin 0.5 pmol/µl
ZipTip C18 Prep
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
ZipTip C18 Prep
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.
ZipTip C18 Prep in PBS/Urea/NaCl
Standard Prep in PBS/Urea/NaCl
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.
Multiple Sodium Adducts
ZipTip C18 Prep
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.
ZipTip C18 Preparation
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.
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.
Proteomics : Experimental Approach
Proteome indicates the proteins expressed by the genome or tissue
Highly acidic/basic, hydrophobic or membrane proteins from MDLC
Success depends upon:
Handling the Gel and Slices
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 4C 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
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).
Enymatic Digestion – Trypsin
Promega Sequencing Grade Modified Trypsin 10-15 ug/ml in 25 mM NH4 Bicarbonate pH 8.0. Store at -70C 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 37C for 16-24 hours. Convection oven is preferable to heat block.
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 30C. Drying can also be done in a lyophilizer.
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.
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
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.
Ammonium Bicarbonate should be used for MALDI compatibility. Tris is a good alternative. Low concentration can be used, 10mM is sufficient.
Porcine Trypsin Peaks
Bovine Trypsin Peaks
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