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Experiment-4: In-gel digestion of proteins for MS analysis


Experiment-4: In-Gel Digestion of Proteins



To excise protein spots from 1D or 2D SDS-PAGE gels and perform in-gel trypsin digestion.
Often one- or two-dimensional gel electrophoresis is applied for separation of complex mixtures of proteins prior to mass spectrometric analysis. In-gel proteolytic digestion of separated proteins is performed to cleave the protein of interest present within the polyacrylamide matrix. The gel-based techniques increase the dynamic range of analysis since they involve sequential separation of proteins based on molecular weight. It removes low molecular weight salts, buffers and other impurities, and polyacrylamide is user friendly matrix for extraction of proteins from gel. Initially, protein identification was carried out by transferring protein from gel to the nitrocellulose or PVDF membrane for tryptic digestion or for N-terminal sequencing. Subsequently, technological advancement introduced in situ tryptic digestion on acrylamide gels or PVDF membrane for identification of proteins of interest.  Presently, mass spectrometry-based studies has shifted in situ digestion to in-gel digestion, due to high sensitivity. Mass spectrometric identification of the target protein greatly depends on the efficacy of the in-gel digestion process that generates mixture of peptides from the target protein through proteolytic digestion. In-gel digestion is a multi-step procedure, which include spot selection, spot excision, stain removal, reduction, alkylation, proteolytic cleavage and finally extraction of the peptides. 
     Steps involved in In-gel digestion
      1.  Excision of spot of interest
After comprehensive analysis of gel images candidate proteins are selected based on which proteins are thought to be upregulated or downregulated as compared to their controls or they may be selected randomly for the study purpose to reveal the protein identity.  The spots of interest are excised for further processing. Spot excision can be performed both in automated (using spot pickers) and manual way. In case of traditional manual approach, the selected spots are excised using sterile scalpel under sterile dust-free environment and kept in sterile microcentifuge tubes.




Fig 1. a) Cleaning of glass plate, b) Spot excision, c) Spot slicing, d) Transfer gel pieces to micro-centrifuge tube

2. Removal of staining agent : Coomassie Brilliant Blue is widely used for staining in 1D and 2-DE PAGE for visualization of proteins. Stain removal is essential prior to mass spectrometric analysis. Immediately after spot picking, excised gels are washed with a mixture of bicarbonate buffer and acetonitrile for removal of the staining agent. ACN reduces the hydrophobic interaction between protein and the stain, while the ionic solution decreases the ionic interaction between negatively charged dye and positively charged protein.




                                                    Fig 2. a) Solutions required for in-gel digestion, b) Addition of solutions

3. Reduction & alkylation :
After stain removal, the next steps include reduction and alkylation of the protein residues to denature the protein into its primary structure. Treatment of the protein residues with DTT breaks the disulfide bonds completely. However, reformation of disulfide bonds may occur and to prevent that IAA, an alkylating agent, is used. It adds acetoamide group to the sulfhydral group and prevents the disulfide bond formation. The chemical reactions for the same are given below: 

                                Fig 3. Reduction of proteins using DTT, Alkylation of proteins using IAA



      4. Tryptic digestion :
Prior to MS identification, proteins are digested to generate peptides. Several proteolytic enzymes are available. Chymotrypsin, trypsin, pepsin are some of the enzymes used for proteolysis. CNBr is one of the chemical agents that cleave the peptide bond at Methionine residue. Trypsin is most widely used proteolytic enzyme used for protein digestion prior to MS analysis. It breaks the peptide bond at carboxyl terminal of basic amino acids such as arginine and lysine.


Fig 4. Tryptic digestion of protein residues; trypsin recognizes the lysine and arginine resides and cleaves to generate smaller peptide fragments.




5. Extraction of digested proteins:
After overnight incubation, peptides generated through proteolytic digestion are extracted using extraction buffer containing 0.1% FA/TFA in 50% ACN solution. Efficient extraction process is essential to ensure the release of peptides from gel-matrix to solution, which is further subjected to mass spectrometric analysis. 



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