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

Подождите немного. Документ загружается.

2 Liquid Chromatography Techniques160

but to generate media with suitable particle sizes

for a range of applications (Table 2.4).

Tab. 2.4: Ion exchange matrices.

Product Form Mean particle size

MiniBeads Polystyrene/divinyl benzene 3 mm

MonoBeads Polystyrene/divinyl benzene 10 mm

SOURCE 15 Polystyrene/divinyl benzene 15 mm

MiniBeads is a matrix made from polystyrene, with divinyl benzene

as cross-linker, to produce highly spherical (monodispersed), very

small (3 mm), non-porous particles that facilitate micro-preparative or

analytical separations when extremely high resolution is more impor-

tant than high binding capacity or high flow rates.

MonoBeads and SOURCE are matrices made from polystyrene

with divinyl benzene to produce highly spherical (monodispersed),

small (10–15 mm), porous particles

Functional groups The functional groups substituted onto a chroma-

tographic matrix (Table 2.5) determine the charge of an IEX medium

i.e. a positively charged anion exchanger or a negatively charged

cation exchanger.

Tab. 2.5: Functional groups used on ion exchangers.

Anion exchanger Functional group

Quaternary ammonium (Q), strong –O–CH

(CH

)

Diethylaminoethyl (DEAE), weak –O–CH

H(CH

)

Cation exchanger Functional group

Sulfopropyl (SP), strong –O–CH

CHOHCH

OCH

–

Methyl sulfonate (S), strong –O–CH

CHOHCH

OCH

CHOHCH

–

Carboxymethyl (CM), weak –O–CH

COO

–

The terms strong and weak refer to the extent that the ionization

state of the functional groups varies with pH.

Strong ion exchangers show no variation in ion exchange capacity

with change in pH. These exchangers do not take up or lose protons

with changing pH and so have no buffering capacity, remaining fully

The most suitable matrix can

be selected according to the

degree of resolution, binding

capacity and flow rates desired

for the separation.

The terms strong and weak do

not refer to the strength with

which the functional groups

bind the proteins.

2.1 Basic Principles of Important Liquid Chromatography Techniques 161

charged over a broad pH range. Strong ion exchangers include Q

(anionic), S and SP (cationic).

There are several advantages to working with strong ion exchan-

gers:

Development and optimization of separations is

fast and easy since the charge characteristics of

the medium do not change with pH.

The mechanism of interaction is simple since

there are no intermediate forms of charge inter-

action.

Sample loading (binding) capacity is maintained

at high or low pH since there is no loss of charge

from the ion exchanger.

& The majority of proteins have isoelectric points

within the range 5.5 to 7.5, i.e. the first choice

would be to start with a strong anion exchanger

at physiological or slightly basic pH. Tris with a pK

of 8.3 is ideal for buffers with a pH between 7.2

and 9.0.

2.1.1.7 Binding Capacity and Recovery

The capacity of an IEX medium is a quantitative measure of its ability

to take up counter-ions (proteins or other charged molecules). The

total ionic capacity is the number of charged functional groups per

mL medium, a fixed parameter of each medium. Of more practical

relevance is the actual amount of protein which can bind to an IEX

medium, under defined experimental conditions. This is referred to

as the available capacity of a medium for a specific protein. If the

defined conditions include the flow rate at which the medium was

operated, the amount bound is referred to as the dynamic capacity for

the medium. The available and dynamic capacities depend upon the

properties of the protein, the IEX medium and the experimental con-

ditions.

The capacity of an IEX medium will vary according to the molecu-

lar size of the specific protein (which affects its ability to enter all the

pores of the matrix) and its charge/pH relationship (the protein must

carry the correct net charge at a sufficient surface density at the cho-

sen pH). With earlier ion exchange media, larger proteins had limited

access to the functional groups, significantly reducing the binding

capacity. Nowadays, state-of-the-art macroporous polymer-based ion

exchange matrices have exclusion limits for globular proteins in

excess of 110

and are therefore suitable for the majority of biomole-

cule separations. Binding capacities will still vary according to the

In practice, in order to achieve

best possible performance, a

protein load of 10 to 20% of

the dynamic capacity is recom-

mended.

2 Liquid Chromatography Techniques162

molecular size of the proteins. For example, a matrix with a high

degree of small pores will exhibit a higher binding capacity for smal-

ler molecules. Experimental conditions such as pH, ionic strength,

counter-ion, flow rate and temperature should all be considered when

comparing binding capacities of different IEX medium. Modern IEX

media show very low levels of non-specific adsorption so that sample

recovery under suitable separation conditions is very high, typically

between 90–100%.

2.1.2

Reversed Phase Chromatography

The following section on reversed phase chromatography (RPC) is

based on information material included in the GE Handbook

“Reversed Phase Chromatography” and modified to the requirements

of protein pre-fraction in proteome analysis.

In the presence of non-polar solvents proteins are more likely to

lose activity than peptides. The interaction of proteins or polypeptides

with a hydrophobic surface in the presence of organic solvents gener-

ally leads to some loss of tertiary structure, often giving rise to differ-

ent conformational states that may interact differently with an RPC

medium. However, denaturation and consequent loss of activity can

be minimized by returning the protein to conditions that favor the

native structure, as demonstrated by the widespread use of RPC for

large-scale purification of recombinant and synthetic proteins and

peptides, such as insulin and growth hormone.

Adsorption chromatography depends on the chemical interactions

between solute molecules and specifically designed ligands chemi-

cally grafted to a chromatography matrix. Over the years, many differ-

ent types of ligands have been immobilized to chromatography sup-

ports for biomolecule separation, exploiting a variety of biochemical

properties ranging from electric charge to biological affinity. An

important addition to the range of adsorption techniques for prepara-

tive chromatography of proteins has been reversed phase chromato-

graphy in which the binding of mobile phase solute to an immobi-

lized n-alkyl hydrocarbon or aromatic ligand occurs via hydrophobic

interaction.

Reversed phase chromatography has found both analytical and pre-

parative applications in the area of biochemical separation and purifi-

cation. Molecules that possess some degree of hydrophobic character,

such as proteins and peptides, can be separated by reversed phase

chromatography with excellent resolution and recovery. In addition,

the use of ion pairing modifiers in the mobile phase allows reversed

phase chromatography of charged solutes such as hydrophilic pep-

tides. Preparative reversed phase chromatography has found applica-

GE Handbook “Reversed Phase

Chromatography, Principles

and Methods”

GE Handbook “Hydrophobic

Interaction and Reversed Phase

Chromatography, Principles

and Methods”

Unless precipitation occurs,

denaturation is not a problem

when using RPC to separate

proteins or peptides for primary

structure determination and

proteome analysis.

2.1 Basic Principles of Important Liquid Chromatography Techniques 163

tions ranging from micropurification of protein fragments for

sequencing by mass spectrometry to process scale purification of

recombinant protein products.

2.1.2.1 Theory and Mechanism of Reversed Phase Chromatography

The actual nature of the hydrophobic binding interaction itself is a

matter of a heated debate, but the conventional wisdom assumes the

binding interaction to be the result of a favorable entropy effect. The

initial mobile phase binding conditions used in reversed phase chro-

matography are primarily aqueous which indicates a high degree of

organized water structure surrounding both the solute molecule and

the immobilized ligand (see Figure 2.4). As solute binds to the immo-

bilized hydrophobic ligand, the hydrophobic area exposed to the sol-

vent is minimized. Therefore, the degree of organized water structure

is diminished with a corresponding favorable increase in system

entropy. In this way, it is advantageous from an energy point of view

for the hydrophobic moieties, i.e. solute and ligand, to associate.

Fig. 2.4: Interaction of a solute with a typical reversed

phase medium. Water adjacent to hydrophobic regions is

postulated to be more highly ordered than the bulk water.

Reversed phase chromatography is an adsorptive process by experi-

mental design, which relies on a partitioning mechanism for small

peptides, in combination with an “on/off” adsorption effect for intact

bigger proteins to accomplish the separation. The solute molecules

partition (i.e. an equilibrium is established) between the mobile

phase and the stationary phase. The distribution of the solute

between the two phases depends on the binding properties of the

medium, the hydrophobicity of the solute and the composition of the

The separation mechanism in

reversed phase chromatography

depends on the hydrophobic

binding interaction between the

solute molecule in the mobile

phase and the immobilized

hydrophobic ligand, i.e. the

stationary phase.

2 Liquid Chromatography Techniques164

mobile phase. Initially, experimental conditions are designed to favor

adsorption of the solute from the mobile phase to the stationary

phase. Subsequently, the mobile phase composition is modified to

favor desorption of the solute from the stationary phase back into the

mobile phase. In this case, adsorption is considered the extreme equi-

librium state where the distribution of solute molecules is essentially

100% in the stationary phase. Conversely, desorption is an extreme

equilibrium state where the solute is essentially 100% distributed in

the mobile phase.

While proteins strongly adsorb to the surface of a reversed phase

matrix under aqueous conditions, they desorb from the matrix within

a very narrow window of organic modifier concentration. Along with

these high-molecular-weight proteins with their unique adsorption

properties, the typical biological sample usually contains a broad mix-

ture of proteins with a correspondingly diverse range of adsorption

affinities. The only practical method for reversed phase separation of

complex biological samples, therefore, is gradient elution. In sum-

mary, separations in reversed phase chromatography depend on the

reversible adsorption/desorption of solute molecules with varying

degrees of hydrophobicity to a hydrophobic stationary phase. The

majority of reversed phase separation experiments are performed in

several fundamental steps.

The first step in the chromatographic process is to equilibrate the

column packed with the reversed phase medium under suitable

initial mobile phase conditions of pH, ionic strength and polarity

(mobile phase hydrophobicity). The polarity of the mobile phase is

controlled by adding organic modifiers such as acetonitrile. Ion-pair-

ing agents, such as trifluoroacetic acid (TFA) or formic acid (FA), are

used in the majority of all protein and peptide separations with RPC.

The polarity of the initial mobile phase (usually referred to as mobile

phase A) must be low enough to dissolve the partially hydrophobic

solute yet high enough to ensure binding of the solute to the reversed

phase chromatographic column.

In the second step, the sample containing the solutes to be sepa-

rated is applied. Ideally, the sample is dissolved in the same mobile

phase used to equilibrate the stationary phase. The sample is applied

to the column at a flow rate where optimum binding will occur.

Once the sample is applied, the chromatographic column is

washed further with mobile phase A in order to remove any unbound

and unwanted solute molecules. Bound solutes are next desorbed

from the reversed phase medium by adjusting the polarity of the

mobile phase so that the bound solute molecules will sequentially

desorb and elute from the column.

In reversed phase chromatography this usually involves decreasing

the polarity of the mobile phase by increasing the percentage of

Reversed phase chromato-

graphy of proteins generally

uses gradient elution instead of

isocratic elution.

2.1 Basic Principles of Important Liquid Chromatography Techniques 165

organic modifier in the mobile phase. This is accomplished by main-

taining a high concentration of organic modifier in the final mobile

phase (mobile phase B). Generally, the pH of the initial and final

mobile phase solutions remains the same.

The gradual decrease in mobile phase polarity (increasing mobile

phase hydrophobicity) is achieved by an increasing linear gradient

from 100% initial mobile phase A containing little or no organic

modifier to 100% (or less) mobile phase B containing a higher con-

centration of organic modifier. The bound solutes desorb from the

reversed phase medium according to their individual hydrophobici-

ties.

The fourth step in the process involves removing substances not

previously desorbed. This is generally accomplished by changing

mobile phase B to near 100% organic modifier in order to ensure

complete removal of all bound substances prior to re-using the col-

umn.

The fifth step is re-equilibration of the chromatographic medium

from 100% mobile phase B back to the initial mobile phase condi-

tions.

The degree of solute molecule binding to the reversed phase med-

ium can be controlled by manipulating the hydrophobic properties of

the initial mobile phase. Although the hydrophobicity of a solute

molecule is difficult to quantitate, the separation of solutes that vary

only slightly in their hydrophobic properties is readily achieved.

Because of its excellent resolving power, reversed phase chromatogra-

phy is an indispensable technique for the high-performance separa-

tion of complex biological samples.

Typically, a reversed phase separation is initially achieved using a

broad range gradient from 100% mobile phase A to 100% mobile

phase B. The amount of organic modifier in both the initial and final

mobile phases can also vary greatly.

& For peptide separations routine percentages of

organic modifier are 5% or less in mobile phase A

and 80% or more in mobile phase B. For intact

protein separations starting with 25% organic

modifier in mobile phase A and 75% or more in

mobile phase B can be generally recommended. In

most cases, acetonitrile is the organic modifier of

choice.

The technique of reversed phase chromatography allows great flexibil-

ity in separation conditions so that the researcher can choose to bind

the solute of interest, allowing the contaminants to pass unretarded

through the column, or to bind the contaminants, allowing the

desired solute to pass freely. Generally, it is more appropriate to bind

Separation in reversed phase

chromatography is due to the

different binding properties of

the solutes present in the

sample as a result of the differ-

ences in their hydrophobic

properties.

2 Liquid Chromatography Techniques166

the solute of interest because the desorbed solute elutes from the

chromatographic medium in a concentrated state. Additionally, since

binding under the initial mobile phase conditions is complete, the

starting concentration of desired solute in the sample solution is not

critical allowing dilute samples to be applied to the column. Ionic

binding may sometimes occur due to ionically charged impurities

immobilized on the reversed phase chromatographic medium, espe-

cially for non- or insufficiently end-capped silica-based media.

The combinations of hydrophobic and ionic binding effects are

referred to as mixed-mode retention behavior. Ionic interactions can

be minimized by judiciously selecting mobile phase conditions and

by choosing reversed phase media with end-capping or based on poly-

mers which are commercially produced with high batch-to-batch

reproducibility and stringent quality control methods.

2.1.2.2 RPC of Peptides, Polypeptides and Proteins

The separation of peptides involves continuous partitioning between

the mobile and stationary phase. Polypeptides and proteins, however,

are too large to partition into the hydrophobic layer; instead, they

adsorb to the hydrophobic phase after entering the column and

remain adsorbed until the concentration of the mobile phase reaches

the critical concentration necessary to cause desorption. They then

adsorb and interact only slightly with the stationary phase as they

elute down the column.

& Desorption takes only place within a very narrow

window of organic modifier concentration. This

results in complete retention until the critical

mobile phase concentration is reached and

sudden desorption of the protein takes place. This

sudden desorption produces sharp peaks.

The “hydrophobic foot” of a polypeptide or protein which is responsi-

ble for the separation, is very sensitive to molecular confirmation. It

can be observed that proteins with intact tertiary structure elute ear-

lier than expected compared to denatured proteins because the

“hydrophobic foot” is involved in the interaction, while the rest of the

protein is in contact with the mobile phase. After desorption very lit-

tle interaction takes place between the protein and the reversed phase

surface and subsequent interactions have little effect on the separa-

tion, i.e. column can be kept short, as column length has little effect

on the separation. Short columns in combination with shallow gradi-

ents can be used very effectively to separate similar proteins. Because

proteins diffuse slowly, RPC results in broader peaks than obtained

with peptides.

2.1 Basic Principles of Important Liquid Chromatography Techniques 167

Column dimensions The adsorption/desorption of proteins respon-

sible for their separation takes place almost entirely near the top of

the column. Therefore, column length does not significantly affect

separation and resolution of proteins. Consequently, short columns

of £ 5 cm in length are often used for protein separation.

Mobile phase considerations

& Since decades, all in all acetonitrile as organic

modifier and TFA as ion-pairing reagent has

proven to be best compromise for reversed phase

chromatography of peptides and proteins. For

direct coupling of LC to mass spectrometry acetic

or formic acid are giving better overall results due

to significant higher ionization efficiency.

Propanol-2 is often used for large or very hydrophobic proteins. The

major drawback of propanol-2 is its high viscosity, especially in mix-

tures with water. To reduce the viscosity of propanol-2, while main-

taining its hydrophobic characteristics, it is recommended using mix-

ture of 50:50 with acetonitrile. Adding 1 to 3% propanol-2 to acetoni-

trile has shown to increase protein recovery in some cases.

Propanol-2 is the best solvent for retaining biological activity. Etha-

nol and methanol are slightly worse. Acetonitrile causes the greatest

loss of biological activity, however, for protein pre-fractionation of pro-

teomic samples this is of no importance, as long as the primary struc-

ture of the molecule remains unaffected and the recovery is suffi-

cient.

In rare cases trifluoroacetic acid (TFA) can be replaced by penta-

fluoropropionic acid (PFPA) or heptafluorobutyric acid (HFBA).

However, the effect is minimal to non-visible.

2.1.3

Affinity Chromatography

Affinity chromatography (AC) separates proteins on the basis of a

reversible interaction between a protein or a group of proteins and a

specific ligand coupled to a chromatography matrix, as illustrated and

described in full detail in the GE Handbook “Affinity Chromatrogra-

phy, Principles and Methods”.

The technique offers high selectivity, hence high resolution, and

usually high capacity for the protein(s) of interest. Enrichment can be

in the order of several thousand-fold and recoveries of active material

are generally very high.

There is no good reason to

deviate from TFA as the ion-

pairing reagent and invest time

and money to evaluate exotic

reagents. RPC coupled to MS

or MS/MS may be seen as an

exception and it is worth trying

other ion pairing reagents to

obtain a better performance in

MS.

2 Liquid Chromatography Techniques168

Affinity chromatography is unique in separation technology since

it is the only technique that enables the purification of a biomolecule

on the basis of its biological function or individual chemical struc-

ture. Purification that would otherwise be time-consuming, difficult

or even impossible using other techniques can often be easily

achieved with affinity chromatography. The technique can be used to

separate active proteins from denatured or functionally different

forms, to isolate, concentrate and enrich pure substances present at

low concentration in large volumes of crude sample and also to

remove specific contaminants, such as the depletion of high-abun-

dant proteins from plasma or serum, prior to proteome analysis.

2.1.3.1 Affinity Chromatography in Brief

Affinity chromatography separates proteins on the basis of a reversi-

ble interaction between a protein or a group of proteins and a specific

ligand coupled to a chromatography matrix.

The technique is ideal for concentration or enrichment steps as

well as for a depletion or elimination in a separation protocol and can

be used whenever a suitable ligand is available for the protein(s) of

interest. With high selectivity, hence high resolution, and high capa-

city for the protein(s) of interest, purification levels in the order of

several thousand-fold with high recovery of active material are achiev-

able.

Biological interactions between ligand and target molecule can be a

result of electrostatic or hydrophobic interactions, and/or hydrogen

bonding. To elute the target molecule from the affinity medium the

interaction can be reversed, either specifically using a competitive

ligand, or non-specifically, by changing the pH, ionic strength or

polarity. Figure 2.5 describes the mode of action of affinity chromato-

graphy. In a single step, affinity purification can offer immense time-

saving over less selective multi-step procedures. The concentrating

effect enables large volumes to be processed. Target molecules can be

separated from complex biological mixtures, native forms can be

separated from denatured forms of the same substance and small

amounts of biological material can be purified from high levels of

contaminating substances.

GE Handbook “Affinity Chro-

matography, Principles and

Methods” (2002) 18-1022-29

2.1 Basic Principles of Important Liquid Chromatography Techniques 169

Fig. 2.5: Schematic representation of the mode of operation of

affinity chromatography and a typical affinity chromatogram.

Successful affinity purification requires a biospecific ligand that can

be covalently attached to a chromatography matrix. The coupled

ligand must retain its specific binding affinity for the target mole-

cules and, after washing away unbound material, the binding

between the ligand and target molecule must be reversible to allow

the target molecules to be removed in an active form. Any component

can be used as a ligand to purify its respective binding partner. Some

typical biological interactions, frequently used in affinity chromato-

graphy for proteomics, are listed below:

Antibody–antigen: for efficient IgG and albumin

depletion from plasma or serum samples;

Lectins: selective glycoprotein enrichment;