Proteomics is a vital and necessary branch of science directed at the in-depth study of protein and their structure to comprehend their function; as an important pharmacological tool in drug discovery and drug development.
The most widely used analytical method of protein parting and quantification, usually consists of integrating protein separation by 2D polyacrylamide gel electrophoresis with micro capillary change phase-liquid chromatography health proteins identification; and finally, diagnosis by mass spectrometry.
However, the occurrence of limitations like the lack of automation and high costs associated within the blend technique led to the study and benefits of a better and more reliable technique involving the use of isotope coded affinity tags (ICAT).
This report talks about the annals of isotope coded affinity tags, its advantages over 2D electrophoretic techniques, the concepts from the approach, its development over time and lastly its application and contribution to the progress and development of analytical research.
It also aspires to touch upon future developmental routes for the technology.
TABLE OF CONTENTS (Hop to)
A. Background
B. Introduction to health proteins quantification
B. 1. 2D Polyacrylamide Gel Electrophoresis
B. 2. Reverse Period High Water Chromatography
B. 3. Mass Spectrometry
B. 4. Problems associated with 2DLC-MS combo technique
C. Introduction to Isotope Coded Affinity Tags (ICAT's)
C. 1. Major advancements in isotope coded affinity label approach
D. Principles of Isotope Coded Affinity Tags (ICAT's)
D. 1. Protein Sampling
D. 2. ICAT reagent - Tagging
D. 3. Peptide Isolation
D. 4. Protein quantification
D. 5. Peptide identification
E. Applications of Isotope Coded Affinity Tags (ICAT's)
E. 1. Applications in the quantitative identification of cancer tumor biomarkers
E. 2. Applications in the quantification of antimalarial drugs and their metabolites in natural fluids
E. 3. Quantification of protein appearance in oxidative-stressed liver organ cells as a therapeutic target for the treating liver disease
E. 4. Quantitative examination of defaulted proteins present in the mind as a healing target for the treatment of brain diseases
E. 5. Applications in the proteomic analysis of recombinant proteins
F. Future Development of Isotope Coded Affinity Tags (ICAT's)
BACKGROUND
Proteins are extremely important components of biologically lively systems plus some of the functions include structural groundwork (connective muscle), transport (carrier protein) 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 connection and function, leading to a little or large interference in the subsequent metabolic pathway within the body due to a variety of reasons; is the major reason behind disease which if not dealt with, can lead to fatality. Because of this, Proteomics is a essential and necessary branch of technology directed at the in-depth research of protein and their buildings; to comprehend their work as an important pharmacological tool in drug discovery and medication development. Improvements in proteomics and genomics through the years through quantitative-structure activity romantic relationship (QSAR) studies and computer aided medicine design (CADD), has helped to recognize novel drugs and their targets to use it.
INTRODUCTION TO Health proteins QUANTIFICATION
The use of Isotope coded affinity tags as a proteins quantification method in proteomics was initially developed in 1999 by Aebersold et al. to aid the diagnosis and purification of recombinant protein[1]. Before the research done in 1999, most widely used approaches to proteins quantification were done by 2D Polyacrylamide Gel Electrophoresis (2D Web page) combined with micro-capillary Liquid reversed period water chromatography (2DLC) and novel electrospray ionization (ESI-MS) or tandem mass spectrometry (MS-MS) way of diagnosis [2].
B. 1. 2D POLYACRYLAMIDE GEL ELECTROPHORESIS
This is basically because 2D Polyacrylamide Gel Electrophoresis (2D Site) is very well known because of its sensitivity and high resolving parting power. Additionally it is a highly versatile technique, and its resourcefulness helps it be highly form after for the parting of biological substances including proteins, based on both physiochemical properties and other chemical-specific relationships. The limit of detection is well recorded to an answer 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 strategy to ensure analytes solubility within sophisticated proteins mixtures.
B. 2. REVERSE PHASE Water CHROMATOGRAPHY
The inclusion of liquid chromatography as another parting step also allows for the further separation of the protein mixtures predicated on difference in retention properties of the components. Recent discovery in the analytical method of liquid chromatography includes the used of two HPLC pumps linked through a detailed 6-port valve system; which results in a more comprehensive parting by gradient elution of sophisticated health proteins mixtures at broadband and quick run times.
B. 3. MASS SPECTROMETRY
Finally, a mass spectrometric approach (Electrospray ionization (EIMS) or tandem mass spectrometry (MSMS)) which gives a UV recognition of proteins and steps the mass to charge ratios of the eluted peptides is employed. The detector produces a thorough chromatogram by plotting UV signals against their corresponding reverse stage retention times, and then the ESI-MS/MS-MS provides mass information for the eluted peptides.
Figure 2: "The development of a 2DLC column and its software with mass spectrometry. (A) A pressure bomb can be used for column packing and sample launching. (B) The circulation rate of in the 2-D column is handled at 100-300 nL/min, and ESI is attained by making use of 2 kV to the gold cable. "[4 & 5]
B. 4. PROBLEMS ASSOCIATED WITH THE 2DLC-MS Combo TECHNIQUE
However, in spite of the attractiveness of the combination technique, lots of limitations are present that makes the technique definately not perfect. It's been documented that complex proteins and peptides with very high alkalinity or basicity plus some trans-membrane proteins can't be separated by this combination method. Also during total cell examination, the combinatorial approach was found to readily accommodate highly abundant protein parting with the lower abundant protein being scarcely detected. The over process also requires several sequential stages including difficult techniques such as in-gel digestion; making the mixture technique highly labour extensive, difficult to automate and therefore non-cost effective. This called for an additional development in proteomic research to overcome these problems by possibly avoiding the parting step by electrophoresis and hence the launch of the use of book Isotope coded affinity tags (ICAT).
INTRODUCTION TO ISOTOPE CODED AFFINITY TAGS
The strategy of isotope coded affinity tagging mainly coupled with a kind of high performance water chromatography and tandem mass spectrometry (LC-MS/MS) is a comparatively new and superior method used in proteomics for the complete quantification and recognition of health proteins sequences within simple or sophisticated protein mixtures. It has been recorded to be simpler as it is with the capacity of straight quantifying the protein from intricate mixtures, eliminating the electrophoretic level. This makes isotope coded affinity tagging more efficient, easily-automated and hence much less labour and cost extensive than the electrophoretic process. The use of ICAT is the new and preferred analytical method for proteins quantification.
Isotope coded affinity tagging is dependant on a class of chemical reagents called 'Isotope coded affinity tags' (ICAT). The ICAT reagent occurs in two forms depending on the quantity of deuteriums; light including none of them or heavy filled with eight. ICAT reagents are made up of three major efficient units:
A particular chemically reactive group responsible for the selective labelling of the SH groups of thiol (cysteine) residues,
An isotope coded linker in charge of the soluble properties of the reagent looked after provides a site for the addition of the isotopic label,
And a biotin affinity tag used to achieve proteins isolation and id. It is determined by the principle of strong binding connection of biotin and avidin.
C 1. MAJOR Progress IN ISOTOPE CODED AFFINITY Label LABELLING
Since the approach was initially presented in 1999 for the labelling of protein mixtures at low levels, there have been valuable technological improvements in the methodology using isotope coded affinity tags (ICAT's) within the pharmaceutical industry. These include:
The design and modification of affinity tags to boost on the chromatographic separation process. [25]
The use of changing peptide specific affinity tags to maximise large-scale quantification on individual processes. [25]
An launch to the blend of different tags to achieve maximum proteome industry [21]
The use of exopeptidases to proficiently take away the affinity tags from the peptides in the purification level [22, 23]
D. PRINCIPLES OF ISOTOPE CODED AFFINITY TAG (ICAT) APPROACH
Isotope coded affinity tags are used for figuring out and quantifying the proteins content of two different cell areas or inhabitants within a mixture. The technique is situated typically on two concepts:
"The peptide sequence of the protein to be quantified (between 5-25 Amino acids long) is made up of sufficient information to recognize that unique health proteins.
And those peptides tagged with the light and heavy reagents respectively are chemically similar and hence serve as very ideal inner specifications for quantification. "
Figure 4. A schematic diagram for the ICAT method of protein quantification.
The rules of Isotope coded affinity tags as noted by Aebersold et al. are split into four levels: Sampling, Tagging, Isolation and Quantification.
D. 1. Proteins SAMPLING
Firstly, two different proteins samples filled with reduced cysteine (thiol) area chains are individually derived; by wearing down the cell composition, and isolating and extracting the protein required from the cell.
D. 2. ICAT REAGENT - TAGGING
For one of the proteins samples, the light form of the ICAT reagent (containing no deuterium) is presented to covalently bind to the SH cysteine residues; whilst for the other, the heavy form of ICAT reagent (filled with eight deuterium) is used. The average person labelled mixtures symbolize different cell says or populations. Both examples are then blended into one complex protein mix and a protease enzyme is put into 'cut-up' or cleave the bigger protein molecules into tagged smaller peptides fragments.
D. 3. PEPTIDE ISOLATION
Avidin is then presented to the combination 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 combination through affinity chromatography. The isolated peptides are then analysed and segregated by micro-capillary high performance water chromatography- mass spectrometry (HPLC-MS/MS).
D. 4. Proteins QUANTIFICATION
This is the most important step of the analytical process as the number and sequence individuality of the protein from which the tagged peptides originated, are automatically decided.
Quantification is achieved by comparing the built-in top intensities for all together eluted pairs of equivalent, doubly incurred peptide ions. The couple corresponds to the two different types of the ICAT reagent with the mass spectrometer jogging successively in two methods. One mode measures the comparative fragmenting of peptides eluting from the micro-capillary column whilst the other files the sequence information of the tagged peptides in the same molar ratios as the matching proteins. This does mean that the chemically similar ICAT-labelled peptide ions are commonly discovered because as they co-elute, they change in mass-to-charge (m/z) proportion 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 series data banking institutions using algorithms and permutations, to identify the protein from which the sequenced peptide originated and hence identify the health proteins. A combination of all results generated on the chromatogram by the mass spectrometer; and examination of the ICAT reagent-labelled peptides therefore can determine the relative quantities as well as the series identities of the the different parts of protein mixtures in one automated procedure.
In mass spectrometry, the ratios between the intensities of the low and top mass components of these pairs of peaks provide an accurate way of measuring the relative abundance of the peptides (and therefore the proteins) in the initial cell pools because the MS power response to confirmed peptide is in addition to the isotopic structure of the ICAT reagents.
E. APPLICATIONS OF ISOTOPE CODED AFFINITY TAGS
The use of ICAT reagent -labelled internal standards, has now turn into a common and fundamental practice in quantitative mass spectrometry. It's been explored to great benefit in several different areas of biochemistry.
E. 1. Quantitative recognition of Cancers biomarkers [9, 10]
Analytical methods that use isotope coded affinity tags are extremely useful and hence popular in the introduction of high throughput method of early cancer detection in humans. [9]The significant quantification and identification of cancer biomarkers using ICAT reagents is a therapeutic target for cancer treatment. In this case, protein samples filled with cancerous and non-cancerous skin cells are denatured and reduced to expose the cysteine -SH peptide residues comprised. They can then eventually labelled with the light or heavy kinds of isotope coded affinity tags in vivo using secure isotopic labelling (SILAC; (e. g. , 2H, 13C, 15N, and 18O)) or in vitro using isobaric tags (iTRAQ). This process allows expressed proteins and peptides in malignant, cancer-derived skin cells to be weighed against non-cancerous skin cells. [8] The use of labelled peptides as inner standards permits relative and/or complete estimation and quantification of the large quantity of the differential proteins present. Emerging solutions like the use of proteins microarrays are opportunities currently being researched and developed for future advancements in cancer tumor biomarker recognition. [10]
E. 2. Quantification of antimalarial drugs and their metabolites in natural fluids [7]
Malaria is a lethal disease responsible for millions of deaths every year, in many exotic 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 composed of proteins, many enzymatic functions are made of peptide - peptide interactions. Isotope coded affinity tagging combined with high performance liquid chromatography has been recorded by 'Kalpesh N. P. et al, 2010' [7] to be a reliable way for the selective persistence and quantification of the powerful antimalarial drugs in biological liquids. ICAT reagents are extremely useful in the removal level of the antimalarial medication from a biological matrix as they provide high peptide selectivity and specificity, to avoid disturbance from multiple antimalarial mix, or endogenous peptides which 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 necessary protein expression in oxidative-stressed liver skin cells as a healing target for the treating liver disease [12]
A major pathogenic event recurrent in several variations of liver organ diseases in humans, includes oxidative stress of the liver organ caused by the forming of reactive oxygen species. Hepatocytes as a rule have mechanisms responsible for the rules of oxidative and anti-oxidative molecules within the cell. However, the occurrence of reactive oxygen kinds in the liver affects major cellular components including cell proteins, and finally, the cell's regulatory capacity. This contributes to metabolic or proliferative liver organ disease and eventual cell fatality. [13]
Reactive oxygen types (ROS) are typically displayed by mitochondria and cytochrome P450 enzymes in liver organ cells. The appearance of certain health proteins molecules referred to as biomarkers within oxidative-stressed liver cells, and their subsequent quantification using ICAT reagents, can enable an early recognition of liver organ disease. It can also allow for the progressive monitoring of liver destruction as a therapeutic target to the treatment of liver disease. [15]
E. 4. Quantitative evaluation of defaulted protein present in the brain as a restorative target for the treating brain diseases.
The brain is an extremely complex structure, essential to the lifestyle of mankind. However, most of the underlying mechanisms responsible for the standard function and mis-function of the brain havent been fully researched. Research into quantitatively characterising the mind proteome and using the examination to comprehend important cell signalling mechanisms [16], is an essential area of neuropoteomics (i. e. proteomic research and development).
The large size use of secure isotope coded affinity tags in quantitative examination of complex brain matrixes has helped to provide internal expectations for relevant peptides that are chemically similar but isotopically different. These internal standards may be used to appropriately identify important biomarkers within the brain just as epilepsy[17]; or absent biomarkers such as the pathogenesis of Parkinson's disease[18].
E. 5. Applications in the proteomic evaluation of recombinant proteins
High-throughput approaches to the quantification and identification of proteins, is broadly applied in the professional synthesis of healing enzymes. [19] Proteomic research on most recombinant proteins, struggle with very low yields and poor solubility which greatly affects the capability to achieve high-throughput health proteins purification. Quantitative methods that use isotope coded affinity tags have been documented to be the only way to attain selective high-throughput protein purification with improved upon produces, solubility and folding of the recombinant health proteins, through the process [19]. This is because, purification processes by biotin affinity normal resulting in great yields of over 90%, so that it is very economically favourable. Combinations of two or more isotopic tags are typically needed to make the most of high-throughput testing. [1]
THE FUTURE OF ISOTOPE CODED AFFINITY TAGS (ICAT's)
The main request portion of isotope coded affinity strategy is in the id of biomarkers as a healing target for disease treatment and reduction. The continuing future of analytical techniques that use Isotope coded affinity tags for peptide-labelling includes:
