Protein quantitation is a technique of "Proteomics" in which abundance of particular proteins is identified in different samples. Proteome dependent on environmental condition is very important. To study particular disease, quantity of protein is required. For example, we took two samples from normal skin cells and the other from squamous carcinoma cells. We diagnose whether protein prevail or not, then we use this protein as disease marker of for the development of therapeutic agent. We may identify how many proteins are present in which stage.
Categories of protein quantitation techniques
The techniques used for protein qunatitation are divided into two categories:
1. 2D-gel Analysis
2. Mass spectrometry technique
1. 2D-gel analysis
In 2D-gel analysis proteins are separated on the basis of size.
Abundance of protein depends on:
First step for the quantitation of protein through 2D-gel is:
Step 1: Image Acquisition
• First step is to digitalize analogue image. Through this we can identify size and shape of protein.
• Image acquisition depends on, which label material is used.
• If we have used radioactive material, we will use X-ray film to map.
• And for the scanning of X-ray film, we will do it through charged coupled devices (CCD) or densitometer.
• If we have used comaise blue/silver staining, to acquire this we will use CCD camera.
• If we have labeled our sample with SYPRO Rubi agent (fluorescently labeled), to acquire the image we will use fluorescence imager. This agent is most commonly used.
• In proteomics techniques, SYPRO Rubi agents are the best labeling materials.
Step 2: Spot Detection
• There are some spots that exist as a discrete entity. Sometimes overlap, sometimes in one line.
• Human eye is very subjective. Our eye's perception varies from person to person, so it's better to do not involve humans in the experiments.
• Before the spot detection, we have to enhance the image. For example, Adobe Photoshop is used for the enhancement of the image i.e. to applying effects to the image.
• Removal of noise (the thing other than spot in gel).
• Image will be emerged with the background through contrast enhancement.
• After noise removal , we have to do edge refinement (identify the boundaries)
• The spots that are in line will be detected.
Step 3: Spot Analysis
• Spot analysis will be done by our self.
• We are getting clear image of spots.
Step 4: Comparison
For the comparison of two gels, we will do through an algorithm called "gel matching". We will find exact abundance in gel matching. For example, we have two gels of age 6 and 12, to identify how many proteins are present in gel of age 6 age and in gel of age 12 , gel matching algorithm is used.
Suppose we know that protein A is present, but now we have to know in which abundance it is present, larger the size of protein more abundant will be the protein. Through this abundance we also come to know the shape of protein.
3. Mass spectrometry technique | ICAT reagents (isotope coded affinity tags)
In this technique, differential labeling of samples for chromatography followed by mass spectrometry, Biotinylated isotopes of iodoacetamide (BII).
• We took two samples from normal cells and cancerous cells.
• Normal cells are labeled with lighter chain of ICAT while cancerous cells are labeled with heavier chain of ICAT. The lighter and heavier forms are used to label different proteins.
• Mixing of normal and cancerous cells, we can identify normal and cancerous cells. As we know that affinity of biotin (A B vitamin that aids in body growth) is with streptavidin (has affinity with cysteine containing polypeptide) and affinity of glucose is with concavalin.
• After mixing of normal and cancerous cells we add trypsin to generate peptide then put it into mass spectrometer.
• The proteins which have cysteine in them, streptavidin bind with it.
• Then labeled proteins were separated and put them into mass spectrometer (for the identification of protein) or autoradiography.
• Then we will analyze the protein.
Drawback of ICAT reagent
If we do not have cysteine in polypeptide then this technique is failed.
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