Lecture 8: Protein Purification and Identification: Part 2

Identifying polypeptides

• why is it important

• Three main methods to identify them

• The most unique feature on a polypeptide chain is the primary structure

• The sequence of amino acids in the polypeptide is derived from the genetic code, BUTmight not match the DNA sequence exactly

• SDS-PAGE and immunoblotting (Western Blotting)

  • Used to confirm the presence of larger polypeptides

  • Antigen-specific antibodies bind to proteins containing specific amino acid sequences or structures

• (Tandem) mass spectrometry (turning it into smaller pieces to deduce what the sequence of the amino acids are, what is present+ their sequence - more complicated)

  • Identify polypeptides after they are ionized and separated into small fragments

  • Computational analysis can be used to determine the sequence based on their mass-to-charge ratio

Immunoblotting (western blotting)

1. Proteins are separated by SDS-PAGE (so they can be removed from the gel and can bind to antibodies) and transferred to a solid-support membrane (blotting).

• side note: however the polymer sheet has high affinity to all proteins (so any thing that is exposed with be bound to this sheet), including antibodies, so not interactions would happen

2. The membrane is exposed to a protein-solution (e.g. milk or BSA) to bind random proteins to any unused regions of the membrane.

• like parking lot with lots of cars, no more parking spots

3. A primary antibody, specific for your POI, is added to recognize linear sequences of amino acids.

• binds to the antibody

4. Secondary antibodies are specific for the Fc domain of the primary antibody and are attached to a fluorescently labeled tag or enzyme that generates a chemi-luminescent, visible product.

• binds to the primary antibody already bound to the antigen, amplifying the signal and helping make it more visible

5. This may show if your POI is present in the sample and its approximate size based on where it migrated on the gel.

• signals will be seem if your POI is present in the gel

Other information

• tubular and actin are loading controls: has the same level fo dark band = same amount of protein eachh time

• To be able to draw conclusions

• Less expression in lysine (maybe some effect more studying need to be done there)

Issue with protein sequence + solution

• Can get the polypeptide sequence from the DNA, however is much more complicated as sequence changes due to post-transcription and translation modifications

• Also polypeptides have 20 amino acids, including different PTMs that need to be identified using just the primary DNA (more complex, labour intensive)

• Solution mass spectrometry

Mass spectrometry

• Peptides are bombarded by a laser to create ionized fragments

• Fragments are attracted to a charged plate detector and analyzed in a mass analyzer by their time of flight

• Time of flight depends on the charge and mass of the molecule

  • More charge = faster

  • Smaller = faster

• Comparison to known peptides can identify the sequence of your polypeptide

Issue: some amino acids might have the exact same atomic mass

Sequencing using Mass spec

• Sequences of peptides are compared to databases to identify protein present.

  • Each protein will be composed a certain amount of atoms that have a particular weight

• If a unique fragment is identified in your sample, it is likely that the protein was present.

  • Fragments of many different sizes

• Protein’s can be digested with a protease before MS produce smaller fragments that are easier to ionize and sequence

• Can also be used to sequence a peptide that is unknown

Process

• protein digestion: will break it into separate parts of the poor in, making git easier to do mass spectroscopy