The aim of this video is to describe the procedure for homogenizing brain tissue to extract proteins for digestion by trypsin and analysis via Mass Spectrometry.
Physical lysis of the tissue is coupled with reagent lysis to achieve the most optimal conditions for identifying as many proteins as possible.
To extract proteins from tissues for mass spectrometric analysis, tissues are first broken open using physical means such as a tissue homogenizer.
Cell lysis is then achieved via chaotropic detergents such as urea, guanidine hydrochloride or thiourea. These chaotropic detergents disturb hydrophobic regions by disrupting hydrogen bonding of amino acids. Sonication is recommended if the sample contains or is contaminated with nucleic acids during homogenization. Remove as much fatty tissue as possible as lipids interfere with the process.
Mass spectrometry compatible detergent such as sodium deoxycholate is also recommended to keep the proteins fully solubilised.
Protein assays may also be performed prior to loading to ensure comparative loading. The spectrohotometric-based Nanodrop may be used to quantitate the proteins.
Following protein solutbiliisation, proteins are first reduced using TCEP (preferred) or DTT, to remove disulfide bonds (cysteine-cysteine bonds) before alkylating with iodoacetamide/iodoacetic acid, to prevent free sulfhydryl groups from reestablishing. The importance of this step is to avoid reformation of cysteine-cysteine bonds once the protein is digested with an endoproteinase such as trypsin.
Extracted proteins are digested into constituent peptides using endoproteinases such as trypsin. This is because peptides ionize and fragment more efficiently than whole proteins.
Salts such as sodium or phosphates and detergents used during the preparation of the sample, must be removed to avoid interference during ionization for mass spec analysis. Some detergents stick to the column or ionize easily, overwhelming the detection but we are not interested in detecting salts or detergents!). Salts and detergents are removed using graphite or C-18 tips or columns.
Concentration of the cleaned peptides via evaporation of the liquid is performed before analysis by ionization. Ionisation is done using most commonly, Electro Spray Ionisation (ESI) followed by orbitrap-based MS analysis.
This video is about protein analysis via mass spectrometry, mass spectrometric analysis of proteins sample preparation, how to prepare samples for mass spectrometry, how to prepare samples for mass spectrometric analysis, preparing samples for proteomics, protein analysis, tissues preparation for proteomics
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//MUSIC
Music: Shine by Joakim Karud
joakimkarud.com/use-my-music/
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// ADWOA
I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. I’ve worked in medical research for years and want to be useful to people new to the lab life. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. Hopefully it helps if you're new to the topic/technique.
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