proteomics quantitative proteomics -...

The identification and quantitation of complex protein mixtures have been facilitated by MS-based quantitative proteomic techniques. Isobaric tag for relative and absolute quantification (iTRAQ) consists of amine-specific, stable isotope reagents that can label peptides of upto eight different biological samples.

Quantitative Proteomics - iTRAQ

In this learning object, the learner will be able to,Describe iTRAQ, and, Recall the Applications of iTRAQ.

Learning Objective

Quantitative Proteomics - iTRAQProteomics

The protein samples to be analyzed are first digested with trypsin into smaller peptide fragments. The trypsin cleaves the proteins at the C-terminal of lysine and arginine residues unless they are followed by a proline residue.

iTRAQ


The peptide fragments generated are separated by SDS-PAGE to simplify the mixture and then tagged with the iTRAQ label. The iTRAQ reagent consists of a reporter group, a balance portion and a peptide reactive group that interacts with the N-terminus of the peptide or free amino group of Lysine residues, giving it an overall mass of 145. The reporter group used to label each peptide sample is unique, with mass varying between 114-117, thereby enabling the labeling and quantification of four samples simultaneously. This has been further improved to allow labeling of eight samples simultaneously.

iTRAQ


The labeled samples are then pooled together. iTRAQ


The pooled samples are purified on a strong cation exchange column to remove any excess unbound iTRAQ reagent. This facilitates sample clean-up prior to further finer separation and purification using reverse phase chromatography.

iTRAQ


Further purification of the SCX purified peptides is carried out by reverse phase liquid chromatography wherein the sample is passed through a column containing a packed stationary phase matrix that selectively adsorbs only certain analyte molecules. The eluted fractions are further characterized by MS.

iTRAQ


The purified labeled peptide fragments are then analyzed by MS/MS. The different masses of the reporter groups allows the peptide fragments to be identified. The reporter group is lost during fragmentation. Relative quantification of up to eight samples can now be performed using iTRAQ.

iTRAQ


Following iTRAQ, the data obtained from tandem mass spectrometry can be analyzed by means of the Mascot search engine. The MS/MS data analysis shareware requires inputs from the user regarding the experimental parameters used such as enzyme cleavage, protein name, modifications, instrument used, peptide charge etc. and the desired search criteria like taxonomy, peptide tolerance etc. Commonly used protein databases against which the MS information is processed to retrieve sequence data include NCBI, MSDB and SwissProt. The data file generated from MS is uploaded and the search carried out.


iTRAQ

A multiple affinity removal system was made use of to carry out immunodepletion of the serum samples from normal controls as well as ovarian cancer patients. This helped in removing the high abundance proteins, leaving behind only the medium and low abundance proteins for iTRAQ analysis.

Application of iTRAQ

The immunodepleted serum samples were then labeled with the iTRAQ reagent and analyzed. The authors detected a total of 220 unique proteins of which 14 were found to be elevated in the ovarian cancer serum samples compared to the healthy controls and four novel candidate biomarkers were detected. Results were validated by Western immunoblotting.



iTRAQ

1. Isobaric Tag for Relative & Absolute Quantification: iTRAQ is a MS based technique for relative and absolute quantification of proteins present in up to four cell preparations by making use of four isobaric, isotope-coded tags that label the proteins via their N-terminal.

2. Protein samples: The samples whose proteins need to be quantified by iTRAQ procedure.

3. Trypsin digestion: Trypsin is a proteolytic enzyme that cleaves proteins at the C-terminal of arginine and lysine residues except when they are followed by proline. This enables large protein samples to be broken down in to small

peptide fragments.

4. Peptide fragments: The smaller fragments obtained upon cleavage of the protein samples after trypsin digestion.

5. iTRAQ label: The iTRAQ reagent consists of a protein reactive group that labels the N-terminus of all peptides as well as free amine groups of lysine side chains, a neutral balance portion and a reporter group, giving it a total mass of 145. The different distribution of isotopes between the reporter and balance groups makes the labels isobaric and enables their detection upon fragmentation and release in MS.


6. SCX purification: Tagged peptides are fractionated on a strong cation exchange column to remove any unbound iTRAQ reagent and to simplify the peptide mixture.

7. LC-MS/MS analysis: The iTRAQ labeled peptides obtained are further purified by reverse phase liquid chromatography and then analyzed by tandem MS. Each tag releases a distinct mass reporter ion upon peptide fragmentation, the ratio of which determines the relative abundances of the peptides.

iTRAQ


1. Normal healthy control: Normal healthy control refers to those who do not have the disease/condition that is being studied. The authors made use of 60 healthy control samples divided into six groups with ten in each.

2. Ovarian cancer patients: Serum samples from 60 patients with serous ovarian carcinoma were divided randomly into six groups, with ten patients in each group.

3. Immunoaffinity depletion: Immunoaffinity depletion is a process that is carried out in order to remove the high abundance proteins present in sera, which hamper the process of detection of medium or low abundance tumour


derived protein markers. In this experiment, the authors made use of three commercial immunoaffinity depletion methods, the multiple affinity removal system (MARS), prior to proteomic analysis.


4. Immunodepleted serum: The serum from which the high abundance proteins have been removed, leaving behind only the medium and low abundance proteins thereby reducing the dynamic range, is known as immunodepleted serum.

proteomics quantitative proteomics -...

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