Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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15.6 Error Tolerance 409

bers of random matches, and it is possible that proteins with a num-

ber of random matches may achieve a significantly high score.

15.4

Charge State

In the case of MALDI all the peptides are singly charged. The choice

is to input the protonated form, the measured mass given in the spec-

trum, or the deprotonated form of the peptide. It is important that

whatever option is chosen, it matches the masses used in the mass

list. If these two pieces of information are different then there is a 1-

Da discrepancy, and this trivializes the performance of the instru-

ment and significantly reduces the discrimination of the database

search.

15.5

Monoisotopic Mass or Average Mass

It is important to select whether the peptide masses are the monoiso-

topic or average masses. If the incorrect option is selected then the

peptide masses have an error of 1 Da, again trivializing the perfor-

mance of the instrument and significantly reducing the discrimina-

tion of the database search.

15.6

Error Tolerance

This option will determine the stringency of the database search and

will be dependant on the mass accuracy of masses measured within

the peptide mass fingerprint. Choose an error tolerance (ppm value)

that allows all the peptides in your peptide mass fingerprint to be

included in the search.

If the error tolerance is too stringent then a peptide mass measure

to a greater error than the tolerance limit contributes nothing towards

the score, even though it may be a peptide from the protein of inter-

est. However, if the error tolerance is too lenient then the number of

spurious matches also increases, reducing the specificity.

Step 15: Preparing for a Database Search410

& Hint: If a 100 ppm error tolerance is selected

then a window of 100 ppm will be applied to all

the theoretical masses in the database, only the

experimental masses which have an error

100 ppm or less will have be accepted by the

search.

Extra information Any additional information may help to constrain

the search, enhancing the discrimination. Thus molecular weight

and pI may be important. It may be useful to remember though that

many proteins may be post-translationally modified or alternatively

spliced, altering the mass and/or the pI.

15.7

Taxonomy

If the origin of the sample is known then the search can be limited to

that particular species or groups of species. This is useful for fully

sequenced and well characterized genomes. However if the species is

not fully sequenced then it may well be useful not to restrict the

search engine to just search against this species. For instance if the

species of origin is mouse, it may also be advantageous to also search

against rat and human; a homologous protein may be matched.

411

Part III

Trouble Shooting

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Additional trouble shooting guides for 2-D electrophoresis are found

in: GE Healthcare Handbook: 2-D Electrophoresis. Principles and

Methods. GE Healthcare Life Sciences (2005); 80-6429-60.

Trouble shooting guides for one-dimensional electrophoresis and

blotting are found in: Westermeier R. Electrophoresis in Practice, 4th

edition. Wiley-VCH, Weinheim (2004).

1.1

Sample Preparation

Symptom Cause Remedy

Cleanup based on

precipitation: pellet

cannot be seen after

centrifugation.

A small protein pellet is

not always easy to see.

Always follow the

instruction stringently

including the direction

of the hinges during cen-

trifugation.

Cleanup based on

precipitation: pellet does

not go back into solution

during resolubilization.

Vortexing has been per-

formed.

Do not vortex! Follow the

hints in Part II, Chapter

1: Sample preparation.

Quantification: the sig-

nal is drifting.

Color reaction influ-

enced by timing.

Follow a well organized

time schedule, as indi-

cated in the instruction.

413

Two-dimensional Electrophoresis

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Including images of bad gels.

1.2

Isoelectric focusing in IPG strips

Symptom Cause Remedy

Rehydration solution is

distributed unevenly

within the gel strip.

Some coating in the strip

holder or reswelling tray.

Wash strip holder and

reswelling tray with

detergent, rinse with de-

ionized water.

Uneven pipetting of the

rehydration solution.

Pipette the solution as

an even streak.

Reswelling tray or IPG-

phor not leveled.

Adjust the level of the

reswelling tray or the

IPGphor on the bench.

Rehydration liquid is left

in the reswelling tray or

strip holder.

Rehydration time too

short.

Rehydrate at least for 6

hours without and 12

hours with sample.

Liquid volume too high. Follow the recommenda-

tions on the package.

IPG strips improperly

stored.

Always store IPG strips

in the freezer, do not

leave them on the bench

at room temperature for

too long a time.

Basic part of the gel

comes off during rehy-

dration.

Surface has been dam-

aged during removal of

cover film.

Always start at the acidic

side to remove the cover

film.

Voltage too low (8 kV or

10 kV not reached).

Short strips (7 cm and

11 cm) are used, 8 kV is

reached in those strips

only with some samples

under exceptional condi-

tions.

Nothing to worry about.

Poor quality of urea and/

or thiourea.

Use high quality urea

and thiourea; remove

ions with a mixed bed

ion exchanger.

Trouble Shooting

414

Symptom Cause Remedy

Too much salt in the

sample.

Remove salts by micro-

dialysis or precipitation;

replace PBS for cell

washing with something

non-ionic like 250

mmol/L sucrose/1

mmol/L Tris.

TCA left in the sample

from precipitation.

Use 10% water/90% ace-

tone for washing instead

of pure acetone.

Bromophenol blue band

stops and does not

migrate completely into

the anode.

Too much salt in the

sample.

See above.

Strip starts to burn at a

certain position.

Too much salt in the

sample.

See above.

Visible brown band

develops.

Tris-chloride in the sam-

ple and rehydration solu-

tion.

Run the sample in the

Manifold.

Cover fluid (paraffin oil)

leaks out of the cup load-

ing strip holder during

IEF.

High protein and salt

load cause water trans-

port that carries the oil

with it.

Reduce the initial voltage

and prolong the first

low-voltage steps. Or,

alternatively use the

Manifold, which is more

tolerant to high protein

and salt concentrations.

The basic part of the

strip swells during IEF

and becomes mechani-

cally unstable.

Many cations in the sam-

ple, for instance Tris.

Avoid adding too much

Tris-base, replace it by

adding 25 mmol/L sper-

mine base, treat the sam-

ple with microdialysis;

apply IEF strips soaked

with deionized water be-

tween gel and electrode

to accommodate ions.

Urea crystallized and

IPG strip dried during

IEF.

Not enough cover fluid

used.

Use 3 mL cover fluid for

18 cm and 24 cm strip

holder and 105 mL for

the manifold.

Cover fluid has been

moved around in the

strip and leaked out.

Reduce the initial voltage

and prolong the first

low-voltage steps.

1 Two-dimensional Electrophoresis

415

Symptom Cause Remedy

Running temperature

was incorrect.

Set the rehydration and

separation temperature

to 20 C.

1.3

SDS PAGE

Symptom Cause Remedy

Gel casting with 14-gel

multicaster: formation of

a gap between gel and

spacer on the side where

the solution is intro-

duced from the bottom.

Contraction of monomer

solution during polymer-

ization in the bottom res-

ervoir.

Pour sufficient displa-

cing solution in the bal-

ance chamber to produce

the thin blue layer in the

entire area of the caster

bottom.

Unequal gel levels in a

multicaster.

Polymerization has

started before solutions

have been leveled out

completely.

Pre-cool the monomer

solution in the refrigera-

tor.

Uneven or curved upper

edge.

Improper overlay. Affini-

ty of overlay solution to

the spacer material.

Spray 0.1% (w/v) SDS

over the edges with a

plant sprayer instead of

overlaying water-saturat-

ed butanol or isopropa-

nol.

Casting gradient gels:

curved upper edge.

Polymerization started at

the bottom, and caused

thermal convection, and

uneven contraction of

the gel.

Make sure that the poly-

merization starts at the

top by adding less cata-

lyst to the dense solu-

tion.

The gel becomes opaque

during polymerization.

The cross-linking factor

is too high.

Check the bisacrylamide

content. Use ready-made

acrylamide/Bis solution.

Upper buffer tank is

leaking.

Hydrostatic balance be-

tween the buffers is not

achieved.

Fill enough buffer into

the lower tank according

to the instruction.

Migration of dye front is

too slow.

Poor quality of reagents

and water.

Make sure that you use

only high quality of

reagents, check the

water.

Trouble Shooting

416

Symptom Cause Remedy

Upper buffer contains

chloride ions.

Do not titrate the run-

ning buffer.

PPA buffer system:

upper and lower buffer

have mixed because of

leakage of upper buffer

tank.

Fill enough buffer into

the lower tank according

to the instruction. Do

not overfill lower and

upper chamber.

The dye front is not

straight in the begin-

ning.

Uneven conductivity

across the IPG strips

originating from the

fixed pH gradient.

Nothing to worry about.

The dye front will

become straight when it

reaches about the middle

of the cassette.

The dye front becomes

curved during the run.

Current leakage in the

ready-made gel cassette.

Remove all excess buffer

or water with the roller.

Close the cassette prop-

erly.

Irregular dye front. Poorly polymerized gels. Optimize catalyst

amounts and use high

quality reagents.

Smiling effect because of

overheating.

Use cooling during the

run and/or reduce the

power setting.

1.4

Staining

Symptom Cause Remedy

Background formed like

a “sail” in the basic part

of the gel.

Complexes between SDS

and basic carrier ampho-

lyte.

Increase fixing time or

destaining time. Try

alternative IPG buffer or

carrier ampholytes.

Silver staining: negative

spots.

Dependent on structure

of protein.

Stain briefly with Coo-

massie blue first.

Silver staining: “donut"-

shaped spots.

Happens with highly

abundant proteins.

Stain briefly with Coo-

massie blue first.

Silver staining: dark

background.

Silver nitrate reduced by

sulfuric compounds, like

softeners.

Use glass or stainless

steel trays only.

1 Two-dimensional Electrophoresis

417

Symptom Cause Remedy

Bad water quality. Check water with a few

drops of silver nitrate so-

lution before you use it

for silver staining.

Sypro Ruby, RuBPS, or

Deep Purple staining:

dye precipitates.

Not sufficiently washed

gel or wrong tray mate-

rial.

Wash the gels after stain-

ing, use only dark poly-

propylene containers.

1.5

DIGE Fluorescence Labeling

Symptom Cause Remedy

The pH of the protein

lysate is less than pH 8

prior to labeling.

The lysis of the cells has

caused a drop in the pH.

1. Increase the buffering

capacity of your lysis

buffer to 40 mmol/L

Tris.

2. Increase the pH of the

lysis buffer by the addi-

tion of a small volume of

50 mmol/L NaOH. Or

add an equal volume of

the lysis buffer that is at

pH 9.5.

The cell wash buffer was

not completely removed

prior to addition of the

lysis buffer.

The samples have been

stored or shipped on dry

ice, CO

has diffused

into the samples, created

carbonic acid.

Seal reagent tubes very

thoroughly with adding

Parafilm



between cup

and cap, and placing

them into several layers

of plastic bags in the dry-

ice box.

The fluorescent signal is

weak when scanned on a

2D gel.

The dyes after reconstitu-

tion have a fixed lifetime

in DMF that may have

been exceeded.

Check the expiry date on

CyDye.

Trouble Shooting

418

Symptom Cause Remedy

The DMF used to recon-

stitute CyDye was of

poor quality or has been

opened for longer than 3

months.

Always use the 99.8%

anhydrous DMF to

reconstitute your CyDye.

Breakdown products of

DMF include amines

which compete with the

protein for the CyDye

labeling

CyDye has been exposed

to light for long periods

of time.

Always store CyDye in

the dark.

CyDye has been left out

of the –20 C freezer for

a long period of time.

Always store CyDye at

–20 C and only remove

them for short periods to

remove a small aliquot.

The pH of the protein

lysate is less than pH 8.

Increase the pH of the

lysis buffer by the addi-

tion of a small volume of

50 mmol/L NaOH. Or

add an equal volume of

the lysis buffer that is at

pH 9.5.

Insufficient mixing of

dye and protein sample.

Click fingers against the

reaction tube during

labeling from the first

moment on.

Problems specifically with

minimal labeling (lysine

labeling).

The fluorescent signal is

weak when scanned on a

2D gel.

Primary amines such as

Pharmalytes or ampho-

lytes are present in the

labeling reaction com-

peting with the protein

for CyDye.

Omit all exogenous pri-

mary amines from the

labeling reaction.

1 Two-dimensional Electrophoresis

419