Sample Cleanup

1

Sample Cleanup
Voyager Training Class
2
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)

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• 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

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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.
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Typical contaminants in protein/peptide
samples No interference TFA, formic acid,
b-mercaptoethanol, DTT, volatile organic
solvents, HCl, NH4OH, acetic acid Tolerable (lt
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
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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

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

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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!
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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.
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• 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

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Micropipets Loaded with Packing Material ZipTip
C18
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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/0.1TFA) minimal volume of 3 µl can be
  used.

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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
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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
Insulin 3.5 pmol/µl
5000
Standard Prep
0
Counts
-5000
ZipTip C18 Prep
-10000
2000
3000
4000
5000
6000
Mass (m/z)
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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)
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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
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Concentration and Buffer Removal of lt0.01 mg/ml
IgG HC by ZipTip C18 Preparation
Analysis of IgG Heavy Chain in 0.2M Tris/ 6M urea
at lt0.01 mg/ml. A concentration effect was seen
as a smaller volume of eluent was used than was
drawn up.
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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.
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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.
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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.
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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.

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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
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In-Gel Digest Fundamentals

• Success depends upon
• Avoiding contamination of samples
• Digesting the protein efficiently
• Maximizing recovery of peptides
• Minimizing losses from handling

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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.

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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 11 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

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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).

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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.

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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.

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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.

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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. 1500 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

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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.
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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 2
163.0570 2273.1600 2289.1649 2530.3339
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Internal Calibration Masses
Other Useful Peaks Internal Calibrants spiked
into sample, e.g. Cal Mix1 _at_ 1500 or 11000
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 1037.526
7 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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