The purpose 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 spectrometry analysis, tissues are first broken up 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 disrupt hydrophobic regions by disrupting the 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 because lipids interfere with the process.
Mass spectrometry compatible detergents such as sodium deoxycholate are also recommended to keep the proteins fully soluble.
Protein testing can also be performed prior to loading to ensure a comparative load. The spectrophotometry-based Nanodrop can be used to quantify the proteins.

After protein solubilization, proteins are first reduced using TCEP (preferred) or DTT, to remove disulfide bonds (cysteine-cysteine bonds) before being alkylated with iodoacetamide/iodoacetic acid, to prevent free sulfhydryl groups from relocating. The importance of this step is to prevent reformation of cysteine-cysteine bonds once the protein has been 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 sample preparation should be removed to avoid interference during ionization for mass specification analysis. Some detergents stick to the column or ionize easily, obscuring 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 carried out before analysis by ionization. Ionization is usually performed using Electro Spray Ionization (ESI), followed by orbittrap-based MS analysis.

This video is about protein analysis via mass spectrometry, mass spectrometry analysis of proteins, sample preparation, how to prepare samples for mass spectrometry, how to prepare samples for mass spectrometry analysis, prepare samples for proteomics, protein analysis, tissue preparation for proteomics

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//MUSIC
Music: Shine by Joakim Karud
http://joakimkarud.com/use-my-music/

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//ADWOA
I am Adwoa (Adwoa Biotech), a graduate in biotechnology. I have worked in medical research for years and want to be helpful to people new to laboratory life. This channel takes you through some of the techniques and concepts I learned while working as a research assistant. Hopefully it helps if you are new to the subject/technique.

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