Proteomics is a vital and necessary branch of science targeted at the in-depth study of proteins and their structure to understand their function; as an important pharmacological tool in drug discovery and drug development.
The most widely used analytical approach to protein separation and quantification, usually involves integrating protein separation by 2D polyacrylamide gel electrophoresis with micro capillary reverse phase-liquid chromatography protein identification; and finally, detection by mass spectrometry.
However, the presence of limitations such as the lack of automation and high costs associated within the combination technique led to the research and introduction of a better and more reliable technique involving the use of isotope coded affinity tags (ICAT).
This report looks at the history of isotope coded affinity tags, its advantages over 2D electrophoretic techniques, the principles associated with the technique, its development over the years and finally its application and contribution to the growth and development of analytical science.
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It also aims to comment on future developmental routes for the technology.
TABLE OF CONTENTS
Section: Section Title Page Number
A. Background 4
B. Introduction to protein quantification 4
B.1. 2D Polyacrylamide Gel Electrophoresis 4
B.2. Reverse Phase High Liquid Chromatography 5
B.3. Mass Spectrometry 5
B.4. Problems associated with 2DLC-MS combination technique 5
C. Introduction to Isotope Coded Affinity Tags (ICAT's) 6
C.1. Major advancements in isotope coded affinity tag approach 6
D Principles of Isotope Coded Affinity Tags (ICAT's) 7
D.1. Protein Sampling 8
D.2. ICAT reagent - Tagging 8
D.3. Peptide Isolation 8
D.4. Protein quantification 8
D.5. Peptide identification 8
E Applications of Isotope Coded Affinity Tags (ICAT's) 9
E.1. Applications in the quantitative identification of cancer
E.2. Applications in the quantification of antimalarial drugs
and their metabolites in biological fluids 9
E.3. Quantification of protein expression in oxidative-stressed liver
cells as a therapeutic target for the treatment of liver disease 10
E.4. Quantitative analysis of defaulted proteins present in the brain as
a therapeutic target for the treatment of brain diseases 10
E.5. Applications in the proteomic analysis of recombinant proteins 10
F Future Development of Isotope Coded Affinity Tags (ICAT's) 11
G References 12
Proteins are very important components of biologically active systems and some of their functions include structural foundation (connective tissue), transportation (carrier proteins) or immunity (antibodies). Specific and selective protein-protein interactions within the body are the basis for key metabolic and kinetic pathways within living organisms. A disruption in a specific protein's interaction and function, leading to a small or large interference in the subsequent metabolic pathway within the body due to any number of reasons; is the major cause of disease which if not dealt with, can lead to fatality. For this reason, Proteomics is a vital and necessary branch of science targeted at the in-depth study of proteins and their structures; to understand their function as an important pharmacological tool in drug discovery and drug development. Developments in proteomics and genomics over the years through quantitative-structure activity relationship (QSAR) studies and computer aided drug design (CADD), has helped to identify novel drugs and their targets for action.
INTRODUCTION TO PROTEIN QUANTIFICATION
The use of Isotope coded affinity tags as a protein quantification method in proteomics was first developed in 1999 by Aebersold et al. to aid the detection and purification of recombinant proteins. Before the research done in 1999, most widely used approaches to protein quantification were done by 2D Polyacrylamide Gel Electrophoresis (2D PAGE) combined with micro-capillary Liquid reversed phase liquid chromatography (2DLC) and novel electrospray ionization (ESI-MS) or tandem mass spectrometry (MS-MS) technique for detection .
Figure 1. "Schematic representation of the procedure for analysis of gel-separated proteins by ESI-MS/MS-MS" 
B.1. 2D POLYACRYLAMIDE GEL ELECTROPHORESIS
This is because 2D Polyacrylamide Gel Electrophoresis (2D PAGE) is very well known for its sensitivity and high resolving separation power. It is also a highly adaptable technique, and its resourcefulness makes it highly sort after for the separation of biological molecules including proteins, based on both physiochemical properties and other chemical-specific interactions. The limit of detection is well documented to a resolution of more than 7000 macromolecules in a singular separation. A large variety and combination of solvents and additives can be used with 2D-PAGE electrophoretic technique to ensure analytes solubility within complex protein mixtures.
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B.2. REVERSE PHASE LIQUID CHROMATOGRAPHY
The inclusion of liquid chromatography as a second separation step also allows for the further separation of the protein mixtures based on difference in retention properties of the components. Recent breakthrough in the analytical approach to liquid chromatography involves the used of two HPLC pumps connected through a detailed 6-port valve system; which results in a more comprehensive separation by gradient elution of complex protein mixtures at high speed and quick run times.
B.3. MASS SPECTROMETRY
Finally, a mass spectrometric technique (Electrospray ionization (EIMS) or tandem mass spectrometry (MSMS)) which provides a UV detection of protein and measures the mass to charge ratios of the eluted peptides is employed. The detector produces a comprehensive chromatogram by plotting UV signals against their corresponding reverse phase retention times, and then the ESI-MS/MS-MS provides mass information for the eluted peptides.
Figure 2: "The construction of a 2DLC column and its interface with mass spectrometry. (A) A pressure bomb is used for column packing and sample loading. (B) The flow rate of in the 2-D column is controlled at 100-300 nL/min, and ESI is achieved by applying 2 kV to the gold wire."[4 & 5]
B.4. PROBLEMS ASSOCIATED WITH THE 2DLC-MS COMBINATION TECHNIQUE
However, in spite of the popularity of the combination technique, a number of limitations exist that makes the technique far from perfect. It has been documented that complex proteins and peptides with very high alkalinity or basicity and some trans-membrane proteins cannot be separated by this combination method. Also during total cell analysis, the combinatorial technique was found to readily accommodate highly abundant protein separation with the lower abundant proteins being scarcely detected. The over process also requires several sequential stages including difficult techniques such as in-gel digestion; making the combination technique highly labour intensive, difficult to automate and hence non-cost effective. This called for a further development in proteomic research to overcome these problems by possibly avoiding the separation step by electrophoresis and hence the introduction of the use of novel Isotope coded affinity tags (ICAT).
INTRODUCTION TO ISOTOPE CODED AFFINITY TAGS
The approach of isotope coded affinity tagging mainly combined with a form of high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) is a relatively new and improved method used in proteomics for the precise quantification and identification of protein sequences within simple or complex protein mixtures. It has been documented to be simpler as it is capable of directly quantifying the proteins from complex mixtures, eliminating the electrophoretic stage. This makes isotope coded affinity tagging more efficient, easily-automated and hence a lot less labour and cost intensive than the electrophoretic process. The use of ICAT is the new and preferred analytical method for protein quantification.
Isotope coded affinity tagging is based on a class of chemical reagents called 'Isotope coded affinity tags' (ICAT). The ICAT reagent occurs in two forms depending on the number of deuteriums; light containing none or heavy containing eight. ICAT reagents are made up of three major functional units:
A distinct chemically reactive group responsible for the selective labelling of the SH groups of thiol (cysteine) residues,
An isotope coded linker responsible for the soluble properties of the reagent and it also provides a site for the addition of the isotopic label,
And a biotin affinity tag used to achieve protein isolation and identification. It depends on the principle of strong binding interaction of biotin and avidin.
Figure 3: "A structure of an ICAT reagent showing the chemically reactive thiol specific group, a linker and a biotin tag.
MAJOR ADVANCEMENTS IN ISOTOPE CODED AFFINITY TAG LABELLING
Since the technique was initially introduced in 1999 for the labelling of protein mixtures at low levels, there have been valuable technological advancements in the approach using isotope coded affinity tags (ICAT's) within the pharmaceutical industry. These include:
The design and modification of affinity tags to improve on the chromatographic separation process. 
The use of variable peptide specific affinity tags to maximise large-scale quantification on individual processes. 
An introduction to the combination of different tags to achieve maximum proteome industry 
The use of exopeptidases to efficiently remove the affinity tags from the peptides in the purification stage [22, 23]
PRINCIPLES OF ISOTOPE CODED AFFINITY TAG (ICAT) APPROACH
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Isotope coded affinity tags are used for identifying and quantifying the protein content of two different cell states or population within a mixture. The technique is based largely on two concepts:
"The peptide sequence of the protein to be quantified (between 5-25 Amino acids long) contains sufficient information to identify that unique protein.
And those peptides tagged with the light and heavy reagents respectively are chemically identical and hence serve as very ideal internal standards for quantification."
Figure 4. A schematic diagram for the ICAT approach to protein quantification.
The principles of Isotope coded affinity tags as documented by Aebersold et al. are divided into four stages: Sampling, Tagging, Isolation and Quantification.
D.1. PROTEIN SAMPLING
Firstly, two different protein samples containing reduced cysteine (thiol) side chains are individually derived; by breaking down the cell structure, and isolating and extracting the proteins required from the cell.
D.2. ICAT REAGENT - TAGGING
For one of the protein samples, the light form of the ICAT reagent (containing zero deuterium) is introduced to covalently bind to the SH cysteine residues; whilst for the other, the heavy form of ICAT reagent (containing eight deuterium) is used. The individual labelled mixtures represent different cell states or populations. The two samples are then combined into one complex protein mixture and a protease enzyme is added to 'cut-up' or cleave the larger protein molecules into tagged smaller peptides fragments.
D.3. PEPTIDE ISOLATION
Avidin is then introduced to the mixture to act as a "magnet" and due to the strong and highly specific binding interaction of biotin and avidin, the ICAT-tagged peptides are isolated from the mixture through affinity chromatography. The isolated peptides are then analysed and separated by micro-capillary high performance liquid chromatography- mass spectrometry (HPLC-MS/MS).
D.4. PROTEIN QUANTIFICATION
This is the most important step of the analytical process as the quantity and sequence identity of the proteins from which the tagged peptides originated, are automatically determined.
Quantification is achieved by comparing the integrated peak intensities for simultaneously eluted pairs of identical, doubly charged peptide ions. The pair corresponds to the two different forms of the ICAT reagent with the mass spectrometer running successively in two modes. One mode measures the comparative fragmenting of peptides eluting from the micro-capillary column whilst the other records the sequence information of the tagged peptides in the same molar ratios as the corresponding proteins. This also means that the chemically identical ICAT-labelled peptide ions are readily identified because as they co-elute, they differ in mass-to-charge (m/z) ratio because of an 8 deuterium difference in the mass of the ICAT-reagents.
D.5. PEPTIDE IDENTIFICATION
The final stage of isotope coded affinity tagging involves an automated correlation with protein sequence data banks using algorithms and permutations, to identify the protein from which the sequenced peptide originated and hence identify the protein. A combination of all results generated on the chromatogram by the mass spectrometer; and analysis of the ICAT reagent-labelled peptides therefore determines the relative quantities as well as the sequence identities of the components of protein mixtures in a single automated operation.
In mass spectrometry, the ratios between the intensities of the lower and upper mass components of these pairs of peaks provide an accurate measure of the relative abundance of the peptides (and hence the proteins) in the original cell pools because the MS intensity response to a given peptide is independent of the isotopic composition of the ICAT reagents.
APPLICATIONS OF ISOTOPE CODED AFFINITY TAGS
The use of ICAT reagent -labelled internal standards, has now become a common and fundamental practice in quantitative mass spectrometry. It has been researched to great advantage in a number of different fields of biochemistry.
E.1. Quantitative identification of Cancer biomarkers [9,10]
Figure 5. "Outline of cancer biomarker discovery through quantitative proteomics."
Analytical methods that employ isotope coded affinity tags are very useful and hence popular in the development of high throughput approach to early cancer detection in humans. The significant quantification and identification of cancer biomarkers using ICAT reagents is a therapeutic target for cancer treatment. In this case, protein samples containing cancerous and non-cancerous cells are denatured and reduced to expose the cysteine -SH peptide residues contained. They can then subsequently labelled with the light or heavy forms of isotope coded affinity tags in vivo using stable isotopic labelling (SILAC; (e.g., 2H, 13C, 15N, and 18O)) or in vitro using isobaric tags (iTRAQ). This approach allows expressed proteins and peptides in malignant, cancer-derived cells to be compared with non-cancerous cells. The use of labelled peptides as internal standards allows for relative and/or absolute estimation and quantification of the abundance of the differential proteins present. Emerging technologies such as the use of protein microarrays are opportunities presently being researched and developed for future improvements in cancer biomarker identification. 
E.2. Quantification of antimalarial drugs and their metabolites in biological fluids 
Malaria is a deadly disease responsible for millions of deaths every year, in many tropical and developing countries. Antimalarial drugs such as chloroquine, mefloquine and pyrimethamine and their metabolites; interact with specific dihydrofolate enzymatic sites in plasmodium falciparum malaria. Since enzymes are largely made up of proteins, many enzymatic functions are made up of peptide - peptide interactions. Isotope coded affinity tagging combined with high performance liquid chromatography has been documented by 'Kalpesh N. P. et al, 2010'  to be a reliable method for the selective determination and quantification of these potent antimalarial drugs in biological fluids. ICAT reagents are very useful in the extraction stage of the antimalarial drug from a biological matrix as they provide high peptide selectivity and specificity, to avoid interference from multiple antimalarial combination, or endogenous peptides that exist within the matrix. The use of the ICAT approach has greatly aided research and development into the pharmacokinetics of different antimalarial drugs especially Chloroquine.[7,8]
E.3. Quantification of protein expression in oxidative-stressed liver cells as a therapeutic target for the treatment of liver disease 
A major pathogenic event recurrent in several variations of liver diseases in humans, involves oxidative stress of the liver caused by the formation of reactive oxygen species. Hepatocytes normally have mechanisms responsible for the regulation of oxidative and anti-oxidative molecules within the cell. However, the presence of reactive oxygen species in the liver affects major cellular components including cell proteins, and eventually, the cell's regulatory ability. This leads to metabolic or proliferative liver disease and eventual cell fatality.
Reactive oxygen species (ROS) are largely represented by mitochondria and cytochrome P450 enzymes in liver cells. The expression of certain protein molecules termed as biomarkers within oxidative-stressed liver cells, and their subsequent quantification using ICAT reagents, can enable an early detection of liver disease. It can also allow for the progressive monitoring of liver damage as a therapeutic target to the treatment of liver disease.
E.4. Quantitative analysis of defaulted proteins present in the brain as a therapeutic target for the treatment of brain diseases.
The brain is a very complex structure, vital to the existence of mankind. However, a lot of the underlying mechanisms responsible for the normal function and mis-function of the brain have not been fully researched. Research into quantitatively characterising the human brain proteome and using the analysis to understand important cell signalling mechanisms , is a very important area of neuropoteomics (i.e. proteomic research and development).
The large scale use of stable isotope coded affinity tags in quantitative analysis of complex brain matrixes has helped to provide internal standards for relevant peptides that are chemically similar but isotopically different. These internal standards can be used to correctly identify important biomarkers present in the brain as in epilepsy; or absent biomarkers as in the pathogenesis of Parkinson's disease.
E.5. Applications in the proteomic analysis of recombinant proteins
High-throughput approaches to the quantification and identification of proteins, is widely applied in the industrial synthesis of therapeutic enzymes.  Proteomic analysis on most recombinant proteins, struggle with very low yields and poor solubility which greatly affects the ability to achieve high-throughput protein purification. Quantitative methods that employ isotope coded affinity tags have been documented to be the only way to achieve selective high-throughput protein purification with improved yields, solubility and folding of the recombinant protein, during the process . This is because, purification processes by biotin affinity normal resulting in great yields of over 90%, making it very economically favourable. Combinations of two or more isotopic tags are typically needed to make the most of high-throughput screening.
THE FUTURE OF ISOTOPE CODED AFFINITY TAGS (ICAT's)
The main application area of isotope coded affinity approach is in the identification of biomarkers as a therapeutic target for disease treatment and prevention. The future of analytical techniques that use Isotope coded affinity tags for peptide-labelling includes: