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Sample Cleanup
Voyager Training Class
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.
Compound
Peptides and
proteins
0.1 to 10 pmol/µL
Oligonucleotides
10 to 100 pmol/µL
Polymers
100 pmol/µL
Concentration
Note: highly dilute samples can be concentrated by Speed-Vac
or Solid Phase Extraction.
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)
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
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.
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
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
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).
Drop dialysis cleanup of Enolase
Before
Yeast Enolase
(47 kDa) in 8 M
urea was dialyzed
for 1 hr on a
Millipore
membrane.
After
Cation Exchange Beads
For 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.
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!
Cation exchange bead cleanup of 31-mer
oligonucleotide
Multiple Sodium
Adducts
No cleanup
Counts
1500
1000
After cation exchange
500
8500
9000
9500
10000
Mass (m/z)
10500
11000
The 31-mer was originally in PBS/2M NaCl.
11500
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
Micropipets Loaded with Packing Material: ZipTip C18
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.
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
Standard Preparation vs. ZipTip
C18 for a Peptide Mixture
ACTH 1-17
2.0 pmol/µl
5000
ACTH 18-39
1.5 pmol/µl
Angiotensin
2.0 pmol/µl
ACTH 7-38
3.0 pmol/µl
Insulin
3.5 pmol/µl
Counts
Standard Prep
0
-5000
ZipTip C18 Prep
-10000
2000
3000
4000
Mass (m/z)
5000
6000
Standard preparation vs. ZipTip
C18 for a mixture of small proteins
20000
Insulin
0.5 pmol/µl
10000
Counts
Apomyoglobin
4.0 pmol/µl
Thioredoxin
2.75 pmol/µl
Standard Prep
0
-10000
ZipTip C18 Prep
-20000
6000
8000
10000
12000
Mass (m/z)
14000
16000
18000
Use of ZipTip C18
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
Concentration and Buffer Removal of <0.01
mg/ml IgG HC by ZipTip C18 Preparation
6000
ZipTip C18 Prep
4000
2000
Counts
0
Standard Prep
-2000
-4000
-6000
20000
30000
40000
50000
60000
70000
Mass (m/z)
80000
90000
100000
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 Efficiently Removes
Protein HC Digestion Buffer
ZipTip C18 Prep in PBS/Urea/NaCl
40000
Counts
20000
0
Standard Prep in PBS/Urea/NaCl
-20000
-40000
1000
1500
2000
2500
3000
Mass (m/z)
3500
4000
4500
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.
ZipTip C18 verses Floating Membrane Dialysis
for Salt Removal from a 31mer Oligonucleotide
Multiple Sodium
Adducts
Standard Prep
Counts
1500
ZipTip C18 Prep
1000
500
Dialysis Prep
8500
9000
9500
10000
Mass (m/z)
10500
11000
11500
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.
High Mass Calibrant: Enolase
Enolase 2
Enolase 1
Photoadduct
ZipTip C18 Preparation
Standard Preparation
44000
Mass (m/z)
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.
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.
Proteomics : Experimental Approach
Proteome indicates the proteins expressed by the genome or tissue
1000
1500
2000
Mass (m/z)
Run gel; stain,
scan
Highly
acidic/basic,
hydrophobic
or membrane
proteins from
MDLC
Extract peptides;
mass analyze
Excise spot,
wash, digest
Database search
In-Gel Digest Fundamentals
Success depends upon:
•
•
•
•
Avoiding contamination of samples
Digesting the protein efficiently
Maximizing recovery of peptides
Minimizing losses from handling
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.
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
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).
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-timeuse 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.
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.
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.
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
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.
Internal Calibration Masses
Porcine Trypsin Peaks
Bovine Trypsin Peaks
842.5100
805.4167
1045.5642
906.5050
2211.1046
1153.5741
2283.1807
2163.0570
2299.1756
2273.1600
2239.1359
2289.1649
2807.3000
2530.3339
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