Sample clean-up (MALDI) Removal of buffer, salts, urea, guanidine, EDTA, glycerol, DMSO, detergents - PowerPoint Presentation

Sample clean-up (MALDI) Removal of buffer, salts, urea, guanidine, EDTA, glycerol, DMSO, detergents
Sample clean-up (MALDI) Removal of buffer, salts, urea, guanidine, EDTA, glycerol, DMSO, detergents

Sample clean-up (MALDI) Removal of buffer, salts, urea, guanidine, EDTA, glycerol, DMSO, detergents - Description


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 Download Presentation

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zip sample tip beads sample zip beads tip remove tips matrix add contaminants exchange cation pipette tfa dilution cold

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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.

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