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8 Structure–Function Relationship and Its Significance in Systems Biology Function

8 Structure–Function Relationship and Its Significance in Systems Biology Function

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biological system works; therefore, the most desirable outcome is new functional information. When

proteomics was born in the mid-1990s, everybody

was fascinated with the ability to identify (catalog)

tens, hundreds, and then thousands of proteins

in one analytical experiment. This did not last long,

as we realized that answers are in relative quantitation rather than the presence or absence of

a particular protein. At this point we hit yet another

wall, which was post-translational modifications,

which increased the complexity of proteomic

experiments by at least two orders of magnitude.

New experimental approaches have been proposed

and collectively great progress has been made in

accumulating huge amounts of data. Although

significant steps in the biological interpretation of

such massive data have been made, our knowledge

about how biological systems function is growing at

a disproportionally low rate. Two hurdles in progress here are correlation of protein structure and

function and protein localization and function. The

latter phenomenon is also called protein moonlighting. This brings us to question what a protein

structure represents in defining its biological function and further on how the structure of a protein

defines its physiological function.

What if we assume that similar sequences of

proteins represent similar functions, whereas

different sequences are responsible for different

functions? We will certainly find many examples to

support such assumptions. Let us consider transmembrane domains of receptors that are hydrophobic and have a helical structure to be

accommodated by a hydrophobic environment of

a lipid bilayer. Furthermore, integrins a 1, 2, and 4

have single-pass transmembrane helical domains

that all play the same function: anchoring these

proteins into the cell membrane. They are all close to

the C-terminal end of the polypeptide chain;

however, all of them have a different primary structure (Figure 2.3).

As we know, integrins are responsible for transmitting signals related to numerous functions and

are part of a/b heterodimers.

Chapter 2



Integrin alpha 1


Integrin alpha 2


Integrin alpha 4


Figure 2.3 Amino acid sequences of transmembrane domains of integrins a 1, 2, and 4.

2.9 Protein Folding and

ProteineProtein Interactions

Proteins fold to reach their conformation associated with function. The process of protein folding is

not fully understood; however, we know that most

proteins are folded during or right after synthesis.

Many proteins, although folded properly, need

further processing and help from chaperons to reach

their final functional structure. Many proteins are

maintained unfolded by chaperons as otherwise they

could not be transported outside of the cell. For

example, Escherichia coli developed a specialized Sectranslocase system for post-translational translocation

of proteins.9,10 This system is a complex of the ATPdriven motor protein SecA and the SecYEG protein

functioning as a membrane-embedded translocation

channel. One of the features of this system is that only

unfolded proteins can be translocated, thus they have to

be kept in a translocation-competent state. SecB holdase, which is an export-dedicated molecular chaperon,

prevents proteins to be translocated from folding and

aggregating. Summarizing, if we extract all proteins

from a cell, denaturate, fragment using, for example,

trypsin, and quantitate based on resulting peptides, we

are unable to conclude whether the protein was

unfolded and complexed with a chaperon and will

contribute to the active pool outside of the cell or was

folded and never destined to be exported. Even if we



measure the stoichiometric ratio of the chaperon to

protein, we do not have evidence of their function and

quantitation gives us limited information.

Another example is the presence of flexible regions

of proteins, which may lead to conformational changes

upon self-interactions forming homopolymers or

upon interactions with other proteins.

Proteineprotein interaction may be mediated by

an induced-folding mechanism. This mechanism has

been proposed for disabling the intrinsic antiviral

cellular defense mechanism by HIV-1 Vif protein.11

Vif neutralizes two components of a human antiviral

defense mechanism, APOBEC3G and APOBEC3F, by

engaging them with the cellular protein complex of

EloB, EloC, Cul5, and Rbx2 to promote degradation

via the proteasomal pathway. In this example,

participation of Vif in such a complex determines one

of its many functions.

2.10 Moonlighting of Proteins

Protein moonlighting is a phenomenon acquired

during the evolutionary process when a single

protein performs more than one function, which is

also associated with specific localization for a specific

function. This phenomenon was described for the

first time by Joram Piatigorsky and Graeme Wistow in

the late 1980s12 but gained more attention after

nailing down this term by Constance Jeffery in

1999.13 The first proteins shown to moonlight were

crystalline and other enzymes14; later proteins, such

as receptors, ion channels, chaperons, or structural

proteins,15 expanded this list.

Due to the lack of a systematic experimental

approach, moonlighting properties of proteins have

been found as a result of other studies that did not

directly target the dual functionality of the proteins of

interest. Nevertheless, the number of such a class of

proteins is increasing rapidly, showing that moonlighting proteins appear to be abundant in all kingdoms of life.16 We may speculate that the list of such

proteins is not complete and that future studies will

add more to the list. Moonlighting phenomenon may

also contribute to various diseases. Therefore, while

Chapter 2

interpreting results of proteomic studies, particularly

when the objective of such studies is to connect

changes in expression levels with a function(s) having

a biological effect, protein moonlighting needs to be


If a protein binds other molecules, whether small

molecules, carbohydrates, or other proteins, it may

acquire a new function that can also be associated with

a different localization. It has to be determined whether

or not such a property falls under the moonlighting

phenomenon. It becomes more complicated when the

pool of relatively abundant extracellular proteins

circulating in body fluid is considered. Proteins circulating as complexes with antibodies may not be quantitated properly using an ELISA assay, and MRM-based

quantitation after proteolytic fragmentation may give

different concentrations. Very often extracellular

proteins are considered a homogeneous population of

molecules; in fact, they may represent an array of

functionally different subsets. It is also possible that

only one subset might be relevant as a biomarker,

whether diagnostic or reflecting molecular mechanisms underlying a pathological state.


1. Kyte J, Doolittle RF. A simple method for displaying the

hydropathic character of a protein. J Mol Biol.

1982;157(1):105-132. Epub 1982/05/05.

2. Biswas KM, DeVido DR, Dorsey JG. Evaluation of methods for

measuring amino acid hydrophobicities and interactions. J

Chromatogr A. 2003;1000(1-2):637-655. Epub 2003/07/25.

3. Cserhati T, Szogyi M. Role of hydrophobic and hydrophilic

forces in peptide-protein interaction: New advances. Peptides.

1995;16(1):165-173. Epub 1995/01/01.

4. Rickard EC, Strohl MM, Nielsen RG. Correlation of

electrophoretic mobilities from capillary electrophoresis with

physicochemical properties of proteins and peptides. Anal

Biochem. 1991;197(1):197-207. Epub 1991/08/15.

5. Dunkley TP, Dupree P, Watson RB, Lilley KS. The use of

isotope-coded affinity tags (ICAT) to study organelle

proteomes in Arabidopsis thaliana. Biochem Soc Trans.

2004;32(Pt3):520-523. Epub 2004/05/26.

6. Yi EC, Li XJ, Cooke K, Lee H, Raught B, Page A, et al. Increased

quantitative proteome coverage with (13)C/(12)C-based, acidcleavable isotope-coded affinity tag reagent and modified data

acquisition scheme. Proteomics. 2005;5(2):380-387. Epub






7. Vara E, Arias-Diaz J, Villa N, Hernandez J, Garcia C, Ortiz P,

et al. Beneficial effect of S-adenosylmethionine during both

cold storage and cryopreservation of isolated hepatocytes.

Cryobiology. 1995;32(5):422-427. Epub 1995/10/01.

8. Ahmed HH, El-Aziem SH, Abdel-Wahhab MA. Potential role of

cysteine and methionine in the protection against hormonal

imbalance and mutagenicity induced by furazolidone in

female rats. Toxicology. 2008;243(1-2):31-42. Epub 2007/10/30.

9. Bechtluft P, Kedrov A, Slotboom DJ, Nouwen N, Tans SJ,

Driessen AJ. Tight hydrophobic contacts with the SecB

chaperone prevent folding of substrate proteins. Biochemistry.


10. Driessen AJ, Nouwen N. Protein translocation across the

bacterial cytoplasmic membrane. Annu Rev Biochem.


11. Bergeron JR, Huthoff H, Veselkov DA, Beavil RL, Simpson PJ,

Matthews SJ, et al. The SOCS-box of HIV-1 Vif interacts with

ElonginBC by induced-folding to recruit its Cul5-containing

ubiquitin ligase complex. PLoS Pathogen. 2010;6(6):


12. Wistow GJ, Piatigorsky J. Lens crystallins: The evolution and

expression of proteins for a highly specialized tissue. Annu Rev

Biochem. 1988;57:479-504. Epub 1988/01/01.

13. Jeffery CJ. Moonlighting proteins. Trends Biochem Sci.

1999;24(1):8-11. Epub 1999/03/24.

14. Chen JW, Dodia C, Feinstein SI, Jain MK, Fisher AB. 1-Cys

peroxiredoxin, a bifunctional enzyme with glutathione

peroxidase and phospholipase A2 activities. J Biol Chem.

2000;275(37):28421-28427. Epub 2000/07/14.

15. Kourmouli N, Dialynas G, Petraki C, Pyrpasopoulou A,

Singh PB, Georgatos SD, et al. Binding of heterochromatin

protein 1 to the nuclear envelope is regulated by a soluble

form of tubulin. J Biol Chem. 2001;276(16):13007-13014. Epub


16. Huberts DH, van der Klei IJ. Moonlighting proteins: An

intriguing mode of multitasking. Biochim Biophys Acta.

2010;1803(4):520-525. Epub 2010/02/11.






Anna Bodzon-Kułakowska,* Anna Drabik,*

Przemyslaw Mielczarek,* Filip Sucharski,*

Marek Smoluch,* Piotr Suder* and

Jerzy Silberring*, †


AGH University of Science and Technology, Krakow, Poland

Centre of Polymer and Carbon Materials, Polish Academy of

Sciences, Zabrze, Poland





3.1.1 Introduction 27

3.1.2 Inhibition of Protease Activity 28

3.1.3 Homogenization 29

3.1.4 Cells as Source of Biological Material for

Proteomics 30

3.1.5 Subcellular Compartments: Organellar

Proteomics 34

3.1.6 Crude Protein Extract: What Is the Next Step? 36

3.1.7 Fractionation Based on Size-Exclusion Filters 38

3.1.8 Chromatographic Methods of Protein

Fractionation 39

3.1.9 Peptide Purification 41

3.1.10 Summary 43

Acknowledgments 44

References 44

Proteomic Profiling and Analytical Chemistry. http://dx.doi.org/10.1016/B978-0-444-59378-8.00003-7

Ó 2013 Elsevier B.V. All rights reserved.






3.2.1 Introduction 46

3.2.2 Conventional Capillary Columns 47

3.2.3 Monolithic Columns 48 Silica-Based Monolithic Columns 49 Organic-Based Monolithic Columns 51 Methacrylate-Based Monolithic Columns Styrene-Based Monolithic Columns 52

3.2.4 Summary and Conclusions 54

References 56











Historical Perspective 58

Principle of Ion-Exchange Chromatography 58

Common Types of IEC Stationary Phases 60

Choice of Ion Exchanger (Cation or Anion?) 63

Choice of Strong or Weak Ion Exchanger 64

Buffers in IEC 65

Ion-Exchange Chromatography in Proteomic

Studies 66

References 68



3.4.1 Principles of Isoelectric Focusing (IEF) 69

3.4.2 Sample Preparation Prior to IEF 72

3.4.3 Isoelectric Focusing in Liquid State 73

3.4.4 Immobilized pH gradient IEF 74

3.4.5 Capillary IEF (CIEF) 75

3.4.6 Isoelectric Focusing in Living Organisms 76

3.4.6 Summary 76

References 77






Piotr Suder, Anna Drabik and Anna Bodzo


AGH University of Science and Technology, Krakow, Poland

3.1.1 Introduction

Technological developments in the field of proteomics clearly indicate that a significant increase of

sensitivity, resolution, and mass accuracy of mass

spectrometers is not a “crystal ball” to correct or

compensate for issues associated with sample preparation. These issues include initial sample preparation, such as homogenization of tissues, cell lysis,

sample cleanup, fractionation, and enrichment, all as

we try to maintain optimal preparative conditions.

The latter is of increasing importance in the case of

high-throughput experiments that include hundreds

of samples, each in limited supply, for example,

clinical material.

There is quite extensive literature in the area of

proteomic sample preparation, as well as biotech

companies that provide protocols and commercial

products that allow researchers to rely on their

reproducibility and efficiency in designing profiling

experiments. In certain instances, reference samples

are offered to help normalize experimental samples

at the analytical level. However, it should be kept in

mind that each experiment is in a way unique and

may require minor or major modifications of sample

preparation protocols. It needs to be recognized

that many protocols were established a decade or

more ago when many current products were not





Thus, the question arises at which step is the

major roadblock in advancing proteomics? Is it in

sample preparation techniques, which quite often is

the first dimension of fractionation? The latter step

should accomplish two goals:

a. create a less complex sample, thus allowing more

precise measurements of low abundant proteins

b. clean up sample from impurities such as salts,

lipids, or remaining solid particles

This part of the chapter focuses on the initial steps

of sample preparation, whereas techniques such as

ion-exchange chromatography (IEC), electrophoresis, and liquid chromatography are discussed in

later sections.

3.1.2 Inhibition of Protease Activity

Samples for full unbiased proteomic profiling are

usually very complex, consisting of hundreds or

thousands of proteins and their forms. Samples can

be extracted from the whole organism, tissue, or

single or a mixture of cells, body fluids, or other

noncellular material, for example, physiological

fluids such as urine, saliva, sperm, cerebrospinal

fluid (CSF), or synovial fluid. In other instances it

may be a cell culture supernatant, tumor tissue, or

biologically infected material. Regardless of the

initial form of biological material, a major concern is

to inhibit proteases that can be released during tissue

or cell disintegration. These samples should be

frozen immediately, most preferably in the presence

of a cocktail of protease inhibitors. Standard protocol

calls for supplementing the buffer with a cocktail of

protease inhibitors that target serine, thiol, aspartic,

and metalloproteinases to prevent random protein

degradation. Obviously, this strategy will not work if

the experimental objective is to profile protein

quantity and enzymatic activity, for example, studies

focused on oxidoreductases, hydrolases, and so on.

Under such circumstances (saving the native enzymatic activity), the only way to attempt to control

undesired sample degradation is to maintain a low

temperature, such as by keeping the sample on ice

during subsequent sample preparations, that is,


homogenization. Alternatively, a specified set of

inhibitors may be applied, thus inhibiting unwanted

enzymes while retaining the activity of a desired

family of proteins. Care should be taken due to the

common cross-specificity of inhibitors.

3.1.3 Homogenization

The first step of sample preparation is the

homogenization of multicellular biological material,

lysis of single cell suspension, or clearing of fluid

samples from debris (cellular or other) and contaminants such as lipid particles in plasma and CSF.

Homogeneity or lack thereof may have a profound

impact on the final outcome of the entire proteomic

experiment; therefore, this step should be performed

with caution equal to other steps/methods. One

source of analytical variability in homogenization

that is difficult to measure is degree of tissue

dispersion. Usually, such a procedure is defined by

the time of homogenization and/or number of cycles.

For ultrasonication, a power level, time, and number

of cycles are provided. Completeness of bacteria or

unicellular organisms’ homogenization can be verified by microscopic observations. It is more difficult

to measure homogenization of subcellular compartments of eukaryotic cells. Many devices have been

developed for homogenization, each having specific

characteristics and parameters, thus strengths and

limitations.1 Therefore, investigators should review

options and choose the most appropriate one or

more for their particular experimental design.

Homogenization is usually not selective toward

sample components, and the main goal is to disintegrate the sample physically to release molecular

components. Homogenized samples still contain

debris, in addition to lipids, saccharides, and

metabolites, which all can affect liquid chromatography (LC) and mass spectrometry (MS) separations/

signals, thus proteomic profiling. In the subsequent

step, samples are usually subjected to centrifugation

yielding the top lipid layer, the middle layer of

soluble proteins and other components, and particulate debris sediment at the bottom. This step is




usually not validated beyond measurement of

protein concentration in the middle layer. Methods

of homogenization are summarized in Table 3.1.1.

It would be good practice to increase reproducibility as well as validation of the results by increased

control of the homogenization process by an internal

standard addition. Supplementing the sample by

adding (“spiking”) an internal standard prior to

homogenization is a widely accepted practice in

analytical chemistry/biochemistry. Although the

addition of an internal standard will not control the

efficiency of extraction, it will control how much

soluble protein is lost, for example, due to nonspecific interactions with cellular debris. By using the

“spiked-in” method it is straightforward to calculate

how much protein is lost during preparation. One

caveat in selecting such an internal standard for

proteomic experiments is the choice of protein representing an average characteristic for the entire pool

of proteins. Using bovine serum albumin for samples

of human origin may provide easily distinguishable

spectra and peptides detectable at the femtomolar

level. It is also important not to add too much of an

internal standard as it is desirable to make precise

measurements at low levels.

3.1.4 Cells as Source of Biological

Material for Proteomics

Homogenization of whole tissue will disintegrate

cells, particularly if it is performed in lysis buffer

containing detergents, which help release proteins.

However, many experimental designs seek more

information, such as characterization of cells in the

tissue. The preferred method of characterizing subsets

of cells is flow cytometry and therefore a single cell

suspension would be the material of choice.2 The

advantage of this approach is the possibility of sorting

cells to extract a population of interest. One caveat is

low yield, which is quite often a major problem with

subsequent proteomic profiling. As an alternative, cell

cultures may be used in proteomic profiling experiments. A cell culture that contains only one type of

cells helps in a more straightforward experimental

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