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

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2 Liquid Chromatography Techniques200

2.4.3.1 IEX under Denaturing Conditions

As the most efficient sample extraction takes place under denaturing

conditions it is a natural choice to maintain these conditions also dur-

ing ion-exchange chromatography. Inter and intra protein interac-

tions are widely suppressed and solubility of proteins is improved.

The addition of protein inhibitors reduces protease activities and

maintains the integrity of the sample. Low percentages of organic

modifier (e.g. 6% propanol-2) or the presence of DTT might be advan-

tageous (Wiegand et al. 2002). Ion exchange chromatography (IEX) is

the natural choice for the first dimension in protein pre-fractionation.

Figure 2.21 illustrates the schematic flow path for the IEX separation.

Fig. 2.21: IEX (first dimension) flow path with single sample injection.

Even small IEX columns have a significant loading capacity for high

protein amounts in the range of 5–10 mg/mL for best possible perfor-

mance. The volume loading capacity in principle is unlimited, i.e.

even large volumes of 100 mL and more of diluted sample can be

applied.

Applications of high sample volumes, of samples with high protein

concentrations or samples with high viscosity have to be handled

with care.

& During sample introduction it is recommended to

considerably reduce the flow rate in order not to

exceed the pressure limit of the column.

The adsorbed proteins are eluted from the column with an ascending

salt gradient and collected in a fraction collector. A fraction size

Wiegand G, Parbel A, Seifert

MHJ, Holak TA, Reuter W. Eur

J Biochem 269 (2002) 5046–

5055.

IEX is a retentive technique

where proteins can be effec-

tively concentrated.

2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 201

approximately three times the base peak width of a typical protein

peak is a good initial setting.

& Try the following setup, before wasting time with

buffer development or column testing.

Columns

RESOURCE Q 1 mL (strong anion exchanger, SAX);

or Mono Q 5/5, slightly better resolution;

or Mini Q 4.6/5.

Buffer composition

A: 20 mmol/l Tris, 8 mol/L urea, 6% (v/v) propanol-2, pH 8.5

(HCl).

B: 20 mmol/L Tris, 8 mol/L urea, 6% (v/v) propanol-2, 1 mol/L

NaCl, pH 8.5 (HCl).

Degas both buffers prior to use.

As it is recommended to use denaturing conditions for protein

extraction, it is natural and good practice that the consecutive step

also takes place in the presence of chaotropic agents. In contrast to

electrophoresis thiourea is not suitable in chromatography, because it

makes UV detection troublesome. The use of propanol-2 as an addi-

tive is intended to eliminate unspecific interactions between station-

ary phase and hydrophobic proteins and maintains its solubility.

The flow-through fraction, before the gradient starts, contains (the-

oretically) the basic proteins with a pI > 8.5. Although they do not

bind to the column they are not lost and will be transferred as all the

other fractions to the next dimension.

2.4.3.2 The Tandem IEX Approach

Depending on the origin and composition of the sample the distribu-

tion between acidic, neutral and basic proteins can differ consider-

ably. In order to obtain a preferably even pI distribution of proteins in

the different fractions it might be beneficial to apply the tandem

arrangement of anion- and cation-exchanger. At a given pH, e.g. 8.5,

negatively charged proteins, anions, bind to the anion-exchanger,

while the other proteins will pass the column with no interaction.

While the adsorbed proteins will be concentrated the other proteins

in the flow-through fraction are even more diluted than before sam-

ple application. By connecting an additional cation-exchanger after

the anion-exchanger all proteins, also the positively charged cations

will be adsorbed. Only the species with a net charge of zero, i.e. with

a pI equal or near the pH of the buffer, will not bind to either col-

umn.

Cost effective, try this first.

The industry standard.

Highest possible resolution for

small sample amounts.

Dissolving urea in water takes a

long time; a heated magnetic

stirrer may help to accelerate

this job.

No filtering through

membranes due to the risk of

introducing PEG into the

sample.

Prepare fresh at least once a

week.

With this approach most of the

proteins will be adsorbed and

concentrated, and the number

of proteins in the flow-through

fraction will be significantly

reduced.

2 Liquid Chromatography Techniques202

In principle every suitable HPLC system, preferably bio-inert, that

is capable to control two additional pressure- or motor-actuated six-

port Rheodyne



- or Valco



-valves, can be used. Figure 2.22 illustrates

the schematic flow path.

In general, there is no need for an autosampler for the first dimen-

sion, as in IEX only one sample can be run at a time (a third INV

valve will be needed if the autosampler is skipped). It is more impor-

tant to cover a wide dynamic range of injection volumes and to be

capable of introducing higher volumes (£150 mL) of diluted protein

extracts.

Fig. 2.22: Tandem IEX (first dimension).

Step 1: Sample injection – loading on stacked SAX/SCX columns.

During sample injection both IEX columns are connected in series.

In principle the order of columns does not matter. However, as the

tandem column approach is derived from the single column SAX lay-

out, the SCX column is installed after the SAX column. As it is not

possible, or at least not so easy to compose a buffer that is equally

good for both modes, it has proven to be acceptable to take the same

buffer as for the SAX column, knowing that it is sub-optimal for the

SCX column. After a sufficient long wash period, until both UV and

conductivity have leveled out, both columns will be separated from

each other by the switching of valve 2 (see Figure 2.23).

The flow path, as outlined

below, has been developed on

Ettan LC controlled by

UNICORN



software.

2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 203

Fig. 2.23: Tandem IEX (1st dimension). Step 2: SAX gradient elution.

Now, only the SAX column is in the flow path, while the SCX column

is “parking” in a stand-by position.

In order to achieve a good separation and a minimum of band

broadening at a comparably low flow rate and a shallow salt gradient

will be applied and the eluting proteins are collected preferably in

micro-titer plates, which are directly compatible with the autosampler

for the consecutive 2

dimension RPC separation. After a full cycle,

including washing, regeneration and re-equilibration, until both UV

and conductivity have leveled out again, both valves switch and con-

nect the SCX column in the flow path, while the SAX column

remains in a parking position (see Figure 2.24).

Now, the SCX column is in the flow path, while the SAX column is

idling in a stand-by position. The same gradient as for the SAX col-

umn will be applied and the eluting proteins are collected in the frac-

tion collector. When the cycle is completed, before another sample

can be loaded, it is recommended to remove remaining proteins and

contaminants from the system and the columns by washing accord-

ing to the data sheet supplied with the columns. After washing out

the cleaning reagents quantitatively the system is ready for the next

sample. Figure 2.25 shows two typical chromatograms.

Best separation at a minimum

of band broadening is achieved

at comparably low flow rate

and a shallow gradient.

2 Liquid Chromatography Techniques204

Fig. 2.24: Tandem IEX (1st dimension). Step 3: SCX gradient elution.

Fig. 2.25: Elution profile of the SAX and SCX column, using a

10 mg sample of E. coli in 8 mol/L urea. Please note the good

representation of the expected theoretical ratio between acidic

and basic proteins in the two chromatograms.

In order to cope with the desired productivity it is convenient to have

one system dedicated to the first dimension IEX and another system

with for the 2

dimension RPC separation. Both systems can easily

be controlled from the same computer.

2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 205

2.4.4

Reversed Phase Chromatography and Protein Pre-fractionation

Fundamental information on this topic can be found in a chapter by

Carr and Moritz in the laboratory manual edited by Richard Simpson

(2003).

Reversed phase chromatography is the ideal subsequent step for

any technique that release samples with a high salt concentration,

such as the fractions that have been processed by ion exchange chro-

matography before. The early eluting peaks (flow-through) contain

no or very little salt, whereas the late eluting peaks contain typically

up to 500 mmol/L NaCl (or more) and all together huge amounts of

urea.

All the very hydrophilic, inorganic buffer constituents as well as

the urea do not interact with the stationary phase and pass through

the column, while the proteins of interest bind to the column – espe-

cially under aqueous, acidic conditions – and get concentrated again

in a sharp narrow zone on the top of the column. In contrast to the

separation mechanism of peptides on an RPC column, which is

believed to be a mixed mode between partition and adsorption phe-

nomena, proteins behave more in a “digital” on/off mode, where des-

orption just takes place in a very narrow range of organic modifier

present. This observation implies that column length does not really

matter.

In practice there is no significant difference in resolution between

a 150 and a 50 mm column.

In addition to shorter separation times also the washing and re-

equilibration periods can be minimized in order to achieve a better

overall throughput. Furthermore, the exposure time of proteins with

the stationary phase is shorter, which also yields in a better recovery.

In general, the life time of RPC columns in protein pre-fractiona-

tion is fairly high. This might be due to the fact that the sample has

already passed the IEX column(s) in the first dimension where

unwanted contaminations and impurities have been removed.

Ideally, the trap column is packed with exactly the same RPC media

as the separation column and has the same inner diameter (i.d.), but

can be much shorter (£ 5 mm). Figure 2.26 shows the flow path of a

system that can process all the fractions from a previous IEX or tan-

dem run in a fully automated and unattended way. After RP desalting

and separation the fractions are collected preferably in microtiter

plates, ready for lyophilization or evaporation and tryptic digestion.

In principle every suitable HPLC system, preferably bio-inert or

bio-compatible, that is equipped with an autosampler, fraction collec-

tor and capable to control two additional pressure- or motor-actuated

six-port Rheodyne- or Valco-valves can be used. The detailed flow

Carr CD, Moritz RL. in

Simpson RJ, Ed. Purifying

proteins for proteomics: A

laboratory manual. Cold Spring

Harbor Laboratory Press, Cold

Spring Harbor, New York

(2003) pp 179–208.

An RPC column deals with

such samples in a perfect dual

way, the combination of

desalting and high-performance

separation.

For protein pre-fractionation it

is recommended to select the

shortest column available.

The installation of a so called

trap column on top of the

separation column can further

extend the life time and robust-

ness of the whole setup.

2 Liquid Chromatography Techniques206

path diagrams as outlined below have been operated on Ettan LC con-

trolled by UNICORN.

During step 1 the pump is pumping acidic eluent A that contains

no organic modifier through the sample loop of the autosampler

(A-905) to the trap column. The proteins bind to the top of the col-

umn while salts and urea pass through the column via the conductiv-

ity monitor to waste (W). The conductivity signal confirms to the

operator whether the desalting process was successful (or not). In

this constellation the RPC separation column is by-passed.

Fig. 2.26: RPC (2nd dimension). Step 1: Sample loading and desalting.

During step 2 (see Figure 2.27) both valves switch to the next position

and after a short, but sufficient wash period the gradient elution

starts in order to desorb the proteins. Now the trap column is flown

through in reverse direction, i.e. the proteins are transported on the

shortest possible way – with a minimum of band broadening – to the

connected separation column followed by the UV monitor and the

fraction collector. In order to minimize the gradient delay volume

and to get shortest possible cycle times from sample injection to sam-

ple injection, the whole autosampler with the sample loop is by-

passed.

After washing and re-equilibration the system switches back to

position 1 and the next cycle can start again and again until all the

IEX fractions have been desalted, separated by RPC and collected

(typical examples are shown in Figure 2.28.

2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 207

Fig. 2.27: RPC (2

dimension). Step 2: Sample elution – reversed flow.

Fig. 2.28: A: Stitched SAX/SCX chromatograms.

B: Typical RPC chromatogram of the SAX/SCX flow-through fraction.

The flow-through fraction is a pool from six consecutive injections of a

total of 10 mg of E. coli lysate.

C: typical RPC chromatogram of SAX fraction no. 3,

same conditions as B.

D: typical RPC chromatogram of SCX fraction no. 5,

same conditions as B.

RPC columns: BioBasic4, 1100 mm, trap column, 110 mm,

flow rate 40 mL/min, gradient 25%B to 75%B in 84 min

(blue: 280 nm; magenta: 214 nm).

Please note the excellent peak

shape and resolution of this

intact protein RPC separation.

From: Hoepker HR, Renlund S,

Edblad N, Wadensten H,

Mascher E, strçm, Fenyç D.

Protein pre-fractionation:

Passing phase or forward-

looking approach? HUPO 4th

Annual World Congress,

Munich (2005).

2 Liquid Chromatography Techniques208

Although high-performance columns and media should be preferred,

an acceptable compromise between resolution and recovery should

be envisaged.

There is some evidence that polymer based resins, especially

macro-porous resins, offer superior recovery – compared with wide-

pore silica based stationary phases – in combination with a fairly

good separation power and resolution. Furthermore, polymer-based

columns are resistant to very extreme pH values, a prerequisite for

efficient column cleaning in order to avoid unwanted memory effects

caused by sticky contaminations between the different samples.

Recently, very impressive protein separations on monolytic col-

umns were published. Peak shapes are extremely narrow and run

times are short. Up to now there are no long term data about life-

times and recoveries available; loadability normally is (too) low for the

application of protein pre-fractionation, where a reasonable loading

capacity is required.

Columns The following columns have been used successfully and

can be recommended:

RESOURCE RPC 1 ml (desalting/trap column);

RESOURCE RPC 6 ml (separation column);

SOURCE 5RPC, 4.6 , with custom-packed trap

columns;

BioBasic



4, available in 1.0, 2.1 and 4.6 mm

i.d., including trap columns.

& If, no stationary phase with exactly the same

selectivity is available for custom-packed trap

columns, it might be beneficial to have a trap

column with slightly higher retention

characteristics.

Although, not specifically tested for the purpose of protein pre-frac-

tionation columns from other manufacturers like GRACE-Vydac,

Agilent, Merck, Dionex, etc. are also suitable.

In the column market, especially for RPC, the offering is over-

whelming. If in doubt what product to buy, or before spending/wast-

ing valuable time for column testing it is recommended to screen the

literature; or – even better – ask an experienced colleague in your

neighborhood for good advice.

Eluent composition

A: 0.065% (v/v) TFA, sequential grade, in water.

B: 0.050% (v/v) TFA, sequential grade, in 84% (v/v) acetonitrile and

16% water.

In protein pre-fractionation

recovery should have a higher

priority than resolution.

For micro-scale protein separa-

tions monolytic columns might

be a promising alternative.

Cost-effective, try this combina-

tion first.

Same selectivity as RESOURCE

RPC, resolution comparable to

silica-based material.

Excellent resolution, suspect to

reduced recovery.

Prepare fresh at least once a

week.

These eluents will provide a

smooth and stable baseline

even at low wavelengths (210

to 220 nm) and high sensitivity.

2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 209

& Note: 84% acetonitrile in water is an azeotropic

mixture, i.e. if this mixture evaporates, e.g. in

summer in a non-air conditioned laboratory, the

composition of the mixture remains widely

unchanged (Handbook of Chemistry and Physics

1997).

Column dimensions and loading capacity With complex samples

such as proteins from cell cultures, tissues or even plasma it is

impossible to separate all the proteins. However, it is essential that

individual proteins are not spread over too many fractions. Keeping

the overlap small is of paramount importance, i.e. it is required to

perform the chromatography under high-resolution conditions in

order to avoid peak broadening and distribution of proteins over too

many fractions. According to a simulation it can be achieved that

under ideal conditions an individual protein is present in not more

than three adjoining fractions.

In order to be able to publish large numbers, many column manu-

facturers specify loading capacities as dynamic loading capacity for a

single standard protein. Under such conditions the column is com-

pletely filled with protein. However, such data are not at all useful for

high-performance separations, where overloading must be avoided.

For example, although dynamic capacity is very high (e.g.

10 mg/ml for albumin to 50 mg/ml for insulin) these figures are of

limited use for high-resolution conditions. As a rule of thumb, load-

ing capacity under high-resolution conditions is at least ten times

lower than the dynamic capacity.

When increasing amounts of protein are loaded on an RPC col-

umn, the peak width remains nearly constant. After a certain point

peak width and asymmetry start to increase and retention times

become shorter. The loading limit of a column is normally defined as

the maximum amount of sample that can be chromatographed with

not more than 10% increase in peak width. This point is called the

“overload point”. In practice the overload point will vary for different

samples. If the peaks are distributed over the effective elution range

of the gradient, more sample material can be injected, and each peak

may have a similar loading capacity compared to standard proteins.

While the effective elution range of the gradient reaches for pro-

teins between 25 to 75% of organic modifier only, the effective peak

capacity per column is finite.

Carried forward to the application area of protein pre-fractionation

the following practical loading capacities, as shown in Table 2.11, can

be expected. It can be assumed that the capacity for multi-peak sam-

ples is higher.

Handbook of Chemistry and

Physics, 78

edition, Lide DR

(Ed. in chief) CRC press, Boca

Raton, Florida (1997).

Serious manufactures give

figures for the practical loading

capacity which often is just

10% of the theoretical capa-

city.

The higher the molecular mass

of a protein, the lower the

capacity.