Sample clean-up (MALDI)
• Removal of buffer, salts, urea, guanidine,
EDTA, glycerol, DMSO, detergents
• Dilution
• Washing
• Drop Dialysis
• Cation Exchange
• Zip Tips
Typical contaminants in protein/peptide samples
• No interference: TFA, formic acid, acetic acid, mercaptoethanol, DTT, HCl, NH4OH.
• Tolerable (<50 mM): HEPES, MOPS, Tris,
NH4OAc.
• Minimizing buffer concentrations improves
performance.
• Avoid: Glycerol, sodium azide, DMSO, SDS,
phosphate, NaCl, Urea (> 2M), guanidine (>2M)
Sample Dilution
• The goal is to dilute the contaminants to the
point where they no longer interfere with
the analysis of the sample.
• Requires high enough analyte concentration
in the sample to provide acceptable data
after dilution.
On-Plate Washing
Buffer and salt removal
Dry sample and matrix
Deposit 1 - 2 L cold 0.1% TFA
Leave for 5 - 10 sec, then remove
Detergent contamination
use 5% isopropanol - cold
Cell extract contamination
Use 100% isopropanol- cold
Drop Dialysis
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To remove low molecular weight contaminants.
Fill a 250 mL container with ultra pure water.
Float the membrane on the water (shiny side up).
Place 10 - 25 L of sample solution on the
membrane.
• Add 1 L of AcN to sample spot to increase
surface area,
• Allow to sit for approximately 45 min.
• Remove with pipette, add matrix, spot.
Cation Exchange
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For removal of alkali metal ions.
Prepare resin in NH4+ form according to instructions.
Place app. 0.1 mg of beads on a clear piece of parafilm.
Add 5 L of sample and 5 L matrix to beads to make a
slurry (the slurry should be app. 50% beads).
• Slowly mix up and down with a pipette 10 - 15 times.
• Allow beads to settle for 15 - 30 sec.
• Pipette supernatant onto sample plate.
– Avoid getting beads onto plate.
– Does not work for positively charged species.
Solid Phase Extraction - Zip Tip
• Zip tip - miniature column chromatography.
• Standard C18 zip tips have 0.6 L bed volume.
• Micro C18 zip tips have 0.2 L bed volume better for automation.
• C4 zip tips for clean-up of protein samples.
• Other types available, e.g. metalchelating for
phosphor peptides and cation exchange.
Procedure for Zip Tip Use
• Condition the tip according to instructions.
• Load the sample onto the tip by pipetting 5 - 10
L of sample up and down several times and
discard the liquid.
• Wash with 3 x 10 L 0.1% TFA to remove salts.
• Elute the sample using 30 - 70% AcN or matrix.