Sample Cleanup
1 / 37

Sample Cleanup - PowerPoint PPT Presentation

  • Uploaded on

Sample Cleanup. Voyager Training Class. Compound. Concentration. Peptides and proteins. 0.1 to 10 pmol/µL. Oligonucleotides. 10 to 100 pmol/µL. Polymers. 100 pmol/µL. Sample Dilution/Concentration

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
Download Presentation

PowerPoint Slideshow about 'Sample Cleanup' - zed

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Sample cleanup

Sample Cleanup

Voyager Training Class

Sample cleanup



Peptides and proteins

0.1 to 10 pmol/µL


10 to 100 pmol/µL


100 pmol/µL

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.

Appearance of matrix sample spot
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 cleanup

  • 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 cleanup

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.

Sample cleanup

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.


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

Sample cleanup

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

Sample cleanup

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).

Sample cleanup



Drop dialysis cleanup of Enolase

Yeast Enolase

(47 kDa) in 8 M urea was dialyzed for 1 hr on a Millipore membrane.

Sample cleanup

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.

Sample cleanup

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!

Sample cleanup

Cation exchange bead cleanup of 31-mer oligonucleotide

Multiple Sodium Adducts

No cleanup




After cation exchange









Mass (m/z)

The 31-mer was originally in PBS/2M NaCl.

Sample cleanup

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

Sample cleanup

Procedure for using ZipTip C18


  • 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 c 18
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 c 18 for a peptide mixture
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

Insulin3.5 pmol/µl


Standard Prep




ZipTip C18 Prep







Mass (m/z)

Standard preparation vs ziptip c 18 for a mixture of small proteins
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



Standard Prep




ZipTip C18 Prep









Mass (m/z)

Use of ziptip c 18
Use of ZipTipC18

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 c 18 preparation


ZipTip C18 Prep




Standard Prep














Mass (m/z)

Concentration and Buffer Removal of <0.01 mg/ml IgG HC by ZipTip C18 Preparation

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 c 18 efficiently removes protein hc digestion buffer

ZipTip C18 Prep in PBS/Urea/NaCl





Standard Prep in PBS/Urea/NaCl











Mass (m/z)

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.

Ziptip c 18 verses floating membrane dialysis for salt removal from a 31mer oligonucleotide

Standard Prep

Multiple Sodium Adducts


ZipTip C18 Prep




Dialysis Prep








Mass (m/z)

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.

High mass calibrant enolase
High Mass Calibrant: Enolase

Enolase 2

Enolase 1


ZipTip C18 Preparation

Standard Preparation

Mass (m/z)



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 c 181
Use of the ZipTipC18


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.

Sample cleanup

Run gel; stain,


Proteomics : Experimental Approach

Proteome indicates the proteins expressed by the genome or tissue




Mass (m/z)

Extract peptides;

mass analyze

Highly acidic/basic, hydrophobic or membrane proteins from MDLC

Excise spot,

wash, digest

Database search

In gel digest fundamentals
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
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 method1
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 method2
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 method3
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.

In gel digest method4
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 method5
In-Gel Digest Method


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 method6
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
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
Internal Calibration Masses

Porcine Trypsin Peaks








Bovine Trypsin Peaks








Internal calibration masses1
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