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

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151

Liquid Chromatography Techniques

This chapter gives inspiration, hints and ideas on how to tackle the

challenges in proteomics by applying the tools of liquid chromatogra-

phy (LC), a complementary approach to the already well established

gel-based workflow.

It is obvious that the capabilities of liquid chromatography are

investigated (Link et al. 1999). There is evidence that LC-based meth-

ods can close some of the gaps and deliver supplementary informa-

tion if we state that 2D electrophoresis is the established standard

method. However, we have always to keep in mind that protein

separation is not just a tool by itself rather than an important inter-

mediate step towards protein identification and characterization by

mass spectrometry.

2.1

Basic Principles of Important Liquid Chromatography Techniques

Proteins are separated using chromatography techniques according

to differences in their specific properties, as shown in Table 2.1. The

following chapter is based on information for protein purification

and adapted to the requirements of protein pre-fractionation in pro-

teome analysis.

Tab. 2.1: Separation principles in protein chromatography.

Property Technique

Charge Ion exchange chromatography,

chromatofocusing

Size Gel filtration

Hydrophobicity Reversed phase chromatography,

hydrophobic interaction chromatography

Biorecognition (ligand specificity) Affinity chromatography

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

In order to overcome some of

the existing limitations of 2D

electrophoresis the need for

complementary approaches

becomes apparent.

Link AJ, Eng J, Schieltz DM,

Carmack E, Mize GJ, Morris

DR, Garvik BM, Yates JR III.

Nature Biotech 17 (1999)

676–682.

GE Healthacre “Protein

Separations’ Handbook Collec-

tion”

2 Liquid Chromatography Techniques152

Knowing that there exist numerous methods for protein separa-

tion, the following chapters cover only the most relevant LC techni-

ques for protein pre-fractionation.

Besides personal preferences for one or the other chromatography

technique there are more objective selection criteria available (see

Table 2.2). It is not only important to look at the performance of a sin-

gle technique, but also it is even more important to plan and consider

how to best interconnect the methods in order to bypass avoidable

intermediate steps that bare the risk of sample losses that lead to an

unwanted reduction in protein recovery.

Tab. 2.2: Selection criteria of LC methods for protein pre-fractionation

in proteomics.

IEX RPC AC GF

Enrichment High High Very high Dilution

Loadability Very high Medium Very high Low

Peak capacity Medium High N/A Low

Desalting capability N/A Yes N/A Yes

Raw sample compatibility Very good Limited Excellent Good

Running costs (column/ eluents) High/low Moderate/

high

Low/low to

excessive/

high

High/low

Volatile buffer No Yes No Possible

Unless there are meaningful affinity columns or media available

(e.g. high-abundancy protein depletion for serum, selective glyco- or

phospho-protein enrichment) ion exchange chromatography is the

first choice to apply the initial sample.

No matter what sample concentration, volume or amount, IEX

works perfectly at any scale. Columns in all different dimensions

packed with the same identical material are available to enable seam-

less up- or down-scalability. Diluted samples are efficiently concen-

trated and separated with high resolution and reasonable peak capa-

city. The method can easily be performed under native or denaturing

and/or reducing conditions, depending on the requirements of the

sample.

& In proteome analysis, the preservation of

biological activity could be of minor importance,

as long as recovery is high.

Due to its triple functionality RPC concentrates diluted samples and

efficiently releases samples from salt, urea or other additives prior to

Originally developed for the

purpose of protein purification,

these methods are also of para-

mount importance to pre-frac-

tionate and de-complexify

crude samples in proteome

analysis.

Ion exchange chromatography

is the first choice.

Columns for protein separa-

tions are somewhat cost inten-

sive, but with a favorable life

time if handled with care.

2.1 Basic Principles of Important Liquid Chromatography Techniques 153

separating the proteins at highest possible resolution and is therefore

an excellent consecutive step for all salt containing fractions. Further-

more, due to the use of volatile solvents RPC fractions can directly be

evaporated or lyophilized, prior to tryptic digestion, in the same vessel

or microtiter plate without unnecessary sample transfer steps.

Affinity chromatography is one of the most selective and specific

separation techniques. Numerous ligands, both commercial and self-

made, are available to isolate or remove a variety of proteins and pro-

tein classes. Dealing with this subject in detail would go beyond the

aim of this book. Some dedicated proteomics applications, as the

enrichment of phospho- and glyco-proteins or peptides as well as the

depletion of high-abundant plasma proteins are described below.

Gel filtration is probably the most simple to perform LC technique.

But, due to its low loadability, low resolution and peak capacity, in

combination with its diluting rather than concentrating character it is

not one of the first methods to consider in proteome analysis. Never-

theless, under special conditions it might be worth to consider.

2.1.1

Ion Exchange Chromatography

Ion exchange chromatography (IEX) for the separation of proteins

was introduced in the 1960s and continues to play a major role in the

separation of proteins. Today, IEX is one of the most frequently used

techniques for the separation of proteins and peptides offering high

resolution and loading capacity. The technique is capable of separat-

ing molecular species that have only minor differences in their

charge properties, for example two proteins differing by one charged

amino acid. These features make IEX well suited for concentration,

enrichment, isolation and fractionation.

This chapter provides a general introduction to the theoretical prin-

ciples that underlie every ion exchange separation. An understanding

of these principles will enable the separation power of ion exchange

chromatography to be fully appreciated.

Practical aspects of performing IEX separations related to proteome

analysis are covered in Chapter 2.3.

2.1.1.1 Charge on Proteins

The charge on proteins arises from some of the amino acid side-

chains, as well as the carboxyl and amino termini, bound ions, and

some prosthetic groups.

The charge on amino acid side-chains depends on the pH of the

solution and the pK

of the side-chains. It is also affected by the

microenvironment around a side-chain. We assume the following:

IEX is used from micro-scale

separations and analysis

through to fractionation of

micrograms, milligrams, grams

of sample and more.

IEX separates molecules on the

basis of differences in their net

surface charge.

2 Liquid Chromatography Techniques154

values for ionizable groups on the protein and that the side-

chains will have the same pK

values regardless of their environment

within the protein (Table 2.3). We also assume that the separation is

based on the total net charge of the protein. Therefore, a protein with

a charge of –15 will bind more tightly to an anion exchanger than a

protein with a charge of –10.

Tab. 2.3: pK

values of charged amino acids, C- and N-terminus.

Group pK

Acids

Carboxyl terminus 3.1

Aspartate 4.4

Glutamate 4.4

Cysteine 8.5

Tyrosine 10.0

Bases

Amino terminus 8.0

Lysine 10.0

Arginine 12.0

Histidine 6.5

The charge on these proteins can be calculated. In brief, when the

pH is less than the pK

of a group, the protonated form of the group

predominates. This leaves the acidic side-chains with a charge

approaching 0 and the basic side-chains with a charge approaching a

limiting value of +1. Conversely, when the pH is greater than the pK

of a group, the deprotonated form predominates, giving acidic side-

chains a charge approaching –1 and basic side-chains a charge

approaching 0. The charge on the protein is the sum of the charges

on the individual amino acid side-chains.

Molecules vary considerably in their charge properties and will

exhibit different degrees of interaction with charged chromatography

media according to differences in their overall charge, charge density

and surface charge distribution. The charged groups within a mole-

cule that contribute to the net surface charge possess different pK

values depending on their structure and chemical microenviron-

ment.

Since all molecules with ionizable groups can be titrated, their net

surface charge is highly pH dependent. In the case of proteins, which

are built up of many different amino acids, containing weak acidic

and basic groups.

Each protein has its own unique net charge versus pH relationship

which can be visualized as a titration curve. Their net surface charge

2.1 Basic Principles of Important Liquid Chromatography Techniques 155

will change gradually as the pH of the environment changes as pro-

teins are amphoteric. The titration curve reflects how the overall net

charge of the protein changes according to the pH of the surround-

ings. Figure 2.1 illustrates some theoretical protein titration curves

(these curves can be generated using an isoelectric focusing gel, but

in practice titration curves are rarely used).

Fig. 2.1: Theoretical protein titration curves, showing

how net surface charge varies with pH.

IEX chromatography takes advantage of the fact that the relationship

between net surface charge and pH is unique for a specific protein.

In an IEX separation reversible interactions between charged mole-

cules and oppositely charged IEX media are controlled in order to

favor binding or elution of specific molecules and achieve separation.

However, at a pH above its isoelectric point, a protein will bind to a

positively charged medium (anion exchanger) and, at a pH below its

pI to a negatively charged medium (cation exchanger). In addition to

the ion exchange interaction, other types of binding may occur. These

effects are mainly due to van der Waals forces and non-polar interac-

tions, which occurs frequently with hydrophobic proteins and pep-

tides.

2.1.1.2 Steps in an IEX Separation

An IEX column comprises a matrix of ideally spherical particles sub-

stituted with ionic groups that are negatively (cationic) or positively

(anionic) charged (see Figure 2.2).

The matrix is usually porous to give a high internal surface area. In

small- and micro-scale separations also non-porous media are used.

The medium is filled into a column to form a packed bed. The bed

is then equilibrated with buffer which fills the pores of the matrix

and the space in between the particles.

A protein that has no net

charge at a pH equivalent to its

isoelectric point (pI) will not

interact with a charged

medium.

2 Liquid Chromatography Techniques156

Fig. 2.2: Principles of an anion exchange separation.

2.1 Basic Principles of Important Liquid Chromatography Techniques 157

The pH and ionic strength as well as the other composition of the

equilibration buffer are selected to ensure that, when sample is

loaded, proteins of interest bind to the column. The proteins which

bind are effectively concentrated onto the column while proteins that

do not have the correct surface charge pass through the column with

the flow of buffer, eluting during or just after sample application.

& Note: The condition of the sample is very

important in order to achieve the most effective

high-resolution separations and make the most

of the high loading capacity. Ideally, samples

should be in the same condition as the starting

buffer.

When the entire sample has been loaded and the column washed so

that all non-binding proteins have passed through the column (i.e.

the UV signal has returned to baseline), conditions are altered in

order to elute the bound proteins.

As ionic strength increases, the salt ions (typically Na

or Cl

–

) com-

pete with the bound proteins for charges on the surface of the med-

ium and one or more of the bound species begin to elute and move

down the column. The proteins with the lowest net charge at the

selected pH will be the first ones eluted from the column as ionic

strength increases. Similarly, the proteins with the highest charge at

a certain pH will be most strongly retained and will be eluted last.

By controlling changes in ionic strength using different forms of

gradient, proteins are eluted differentially in a purified, concentrated

form. A wash step with very high ionic strength buffer removes most

tightly bound proteins and impurities at the end of an elution. The

column is then re-equilibrated in start buffer before applying another

sample in the next run.

2.1.1.3 Selectivity

Good selectivity (the degree of separation between peaks) is a more

important factor than high efficiency in determining resolution (see

Figure 2.3) and depends not only on the nature and number of the

functional groups on the matrix, but also on the experimental condi-

tions, such as pH (influencing the protein charge), ionic strength and

elution conditions. It is the ease and predictability with which these

experimental conditions can be manipulated, when using a suitably

designed chromatography medium, which gives IEX the potential of

extremely high resolution.

Most frequently, proteins are

eluted by increasing the ionic

strength (salt concentration) of

the buffer.

The higher the net charge of the

protein, the higher the ionic

strength that is needed for

elution.

2 Liquid Chromatography Techniques158

Fig. 2.3: Effect of selectivity and efficiency on resolution.

From GE Healthcare handbook: “Hydrophobic Interaction

and Reversed Phase Chromatography. Principles and Methods”.

2.1.1.4 Selectivity and pH

In protein separation good selectivity is achieved by performing IEX

separations at pH values carefully selected to maximize the differ-

ences in net charge of the components of interest. Optimum selectiv-

ity can be expected at a pH where there is maximum separation

between the titration curves for the individual proteins (i.e. the differ-

ence in net charges between the species is greatest) and when using

an ion exchanger with a charge opposite to the charge of the proteins

at the particular pH. The order in which proteins are eluted cannot

always be predicted with absolute certainty since a titration curve

(produced in practice by measuring electrophoretic mobility in a gel)

reflects the total net charge on a protein and IEX chromatography

depends on the net charge on the surface of the protein.

For the purpose of protein pre-fractionation where in principle all

proteins must be isolated, which in practice is impossible, a pH is

selected with regard to protein stability and recovery for all, or at least

the majority of the proteins.

2.1.1.5 Selectivity and Elution

The figures below illustrate the most common forms of IEX separa-

tion in which proteins are eluted by increasing the ionic strength of a

buffer (typically with NaCl) using linear gradient or step elution. The

UV absorbance and conductivity traces show the elution of protein

peaks and the changes in salt concentration, respectively, during elu-

tion.

A pH interval of plus/minus

1.5 pH unit apart from the

physiological pH seems to be

reasonable.

2.1 Basic Principles of Important Liquid Chromatography Techniques 159

Buffer volumes used during sample application, elution, washing

and re-equilibration are expressed in column volumes, for example 5

CV=5 mL for a column with a 1 mL bed volume.

Using column volumes to describe a separation profile facilitates

method development and transfer of methods to columns of different

dimensions when scaling-up or scaling-down.

Gradient elution is used when starting with an unknown sample

(as many components as possible are bound to the column and eluted

differentially to see a total protein profile) and for high-resolution

separation that are required in protein pre-fractionation.

2.1.1.6 Components of Ion Exchange Media

Chromatography media for ion exchange are made from porous or

non-porous matrices, chosen for their physical stability, their chemi-

cal resistance to stringent cleaning conditions and their low level of

non-specific interaction. The matrices are substituted with functional

groups that determine the charge of the medium.

Matrix

High porosity offers a large surface area covered

by charged groups and so ensures a high bind-

ing capacity. High porosity is also an advantage

when separating large proteins. Non-porous

matrices are preferable for extremely high-reso-

lution separations when diffusion effects must

be kept to a minimum.

An inert matrix minimizes non-specific interac-

tions with sample components.

High physical stability ensures that the volume

of the packed medium remains constant despite

extreme changes in salt concentration or pH,

thus improving reproducibility and avoiding the

need to repack columns.

High physical stability and uniformity of particle

size facilitate high flow rates, particularly during

cleaning or re-equilibration steps, to improve

throughput and productivity.

High chemical stability ensures that the matrix

can be cleaned using stringent cleaning solu-

tions if required.

Modern IEX media use polymer-based matrices

to fulfill not only the requirements for high

binding capacity, chemical and physical stability,