Dilution Washing Drop Dialysis Cation Exchange Zip Tips Typical contaminants in proteinpeptide samples No interference TFA formic acid acetic acid mercaptoethanol DTT HCl NH 4 ID: 756048
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Slide1
Sample clean-up (MALDI)
Removal of buffer, salts, urea, guanidine, EDTA, glycerol, DMSO, detergents
Dilution
Washing
Drop Dialysis
Cation
Exchange
Zip TipsSlide2
Typical contaminants in protein/peptide samples
No interference: TFA, formic acid, acetic acid,
-mercaptoethanol, DTT, HCl, NH
4
OH.
Tolerable (<50 mM): HEPES, MOPS, Tris, NH
4
OAc.
Minimizing buffer concentrations improves performance.
Avoid: Glycerol, sodium azide, DMSO, SDS, phosphate, NaCl, Urea (> 2M), guanidine (>2M)Slide3
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.Slide4
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- coldSlide5
Drop Dialysis
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.Slide6
Cation Exchange
For removal of alkali metal ions
.
Prepare resin in NH
4
+
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.Slide7
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.Slide8
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.