Post-translational modifications, extraction and detection of proteins

Learning Objectives for Protein Analysis

• Get familiar with the concept of protein expression
• Understand how proteins can be detected
• Get familiar with the protein analysis and purification methods

Definitions in Molecular Biology

• Genome: The entire set of genes or genetic material present in a cell or organism
• Transcriptome: The entire set of all the mRNA molecules expressed from the genes of a cell or organism
• Proteome: The entire set of proteins that is or can be expressed by a cell or organism

Proteome Complexity and Modifications

36 FUSI جامعة الملك عبد العزيز King Abdulaziz University Post-translational modifications, extraction and detection of proteins Dr. Hossam Tayeb MLT-F-631 2023 hhtayeb@kau.edu.sa

Genome Proteome A Blueprint House Plan Front Door Hall 0 Kitchen Garage Bathroom Living Area Back Door Backyard

Proteome Complexity AAA AAA AAA AAA AAA Genome 20-25,000 genes Transcriptome ~100,000 transcripts Alternative splicing Proteome >1,000,000 proteins Post-translational modifications

Post-translational Modifications (PTM)

• What is PTM? Covalent modification of amino acids of specific proteins
• What is the purpose of PTM?
  1. PTM play a major role in protein folding and stability -+ important role in the cellular functions
  2. Most proteins undergo some modification before undertaking any function assigned to them

Types of PTM

• Covalent changes: Addition of one or more groups of phosphoryl, acetyl or glycosyl to one or more of amino acids in a protein
• Proteolytic cleavage: Cleavage of one or more peptide bonds to remove a portion of a protein by Protease e.g. zymogens: which are inactive forms of enzymes are activated by the removal of some portion of the protein

Ribosome R OH Me 0 Methylation Adds a methyl group, usually at lysine or arginine residues Glycosylation Attaches a sugar, usually to an "N" or "O" in an amino acid side chain O M n Lipidation Attaches a lipid, such as a fatty acid, to a protein chain V Ac 0 Acetylation Adds an acetyl group to an N-terminus of a protein or at lysine residues SUMOylation Adds a small protein SUMO (small ubiquitin-like modifier) to a target protein Su Disulfide Bond Covalently links the "S" atoms of two different cysteine residues 0 ANS-S Ubiquitination Adds ubiquitin to lysine residue of a target protein for degradation Ub Protein Hydroxylation Attaches a hydroxyl group (-OH) to a side chain of a protein Phosphorylation Adds a phosphate to serine, threonine or tyrosine M P

Covalent Changes: Phosphorylation

• Phosphorylation (most common) is an important covalent PTM in cell signalling pathways.
• Over 30% of eukaryotic proteins are subjected to phosphorylation.
• This modification is catalysed by protein kinases.
• Phosphorylation modification may turn "on" or "off" the function of a protein OFF ON P Activation Phosphatase P P ON OFF Inactivation

Proteolytic Cleavage Example

• Cleavage of one or more peptide bonds to remove a portion of a protein by Protease
• E.g. insulin it is released as preproinsulin. After removal of signal sequence it is called proinsulin, which is a further cleaved to remove C Chain to form mature insulin C Chain A Chain A Chain COOH COOH H2N COOH S S S 5 - Ş Ș Ș Ș H2N H2N H_N COOH Signal Peptide B Chain B Chain B Chain Preproinsulin Insulin Preproinsulin C Chain A Chain

Detecting Post-translational Modifications

• Before detecting any PTM, modified proteins need to be synthesized and purified in a detectable limit
• Special purification methods of the post-translational modified proteins are needed
• Special detection methods are also needed to study these proteins
• PTM of proteins can be experimentally detected by a variety of techniques, including immunoprecipitation techniques (e.g. Western blotting), mass spectrometry, in vitro biochemical assays and immunofluorescence

Protein Purification Steps

• Expression and purification processes are important in studying an individual protein, and involve the following three basic steps:
  1. Cell culture
  2. Cell lysis - Lysis buffer > results in cell lysate (expressed proteins)
  3. Purification
     1. Protein binding to a matrix or resin
     2. Washing
     3. Elution

Protein purification Cell culture Produce cell lysate using lysis buffer Immunoprecipitation of Modified proteins using resin (e.g. beads) Wash unbound proteins Elute and collect the protein of interest

Immunoprecipitation Techniques

• A core technique in PTM to detect low traces or abundance for the protein of interest
• Protein of interest is enriched or detected using affinity-based purification technology using targeting or binding molecules, e.g. antibodies
• Example, western blot analysis

Western Blot Analysis

• Proteins are separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)
• SDS: is a detergent used to denature proteins to linearize them
• Polyacrylamide gels are a highly cross-linked gel matrix
• The gel acts as MEDIUM where proteins move through in response to the electric field and molecular weight

Protein samples and marker loaded in vertical SDS-PAGE system Buffer I Buffer - - I I -> + Buffer Direction of migration of samples in vertical SDS-PAGE system SDS-PAGE gel after Coomassie blue staining

Sample loading Cathode Protein mixture Porous gel Anode Direction of anion migration Electrophoresis

Western Blot Protein Separation

• Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge
• By denaturing the proteins and giving them a uniform negative charge, it is possible to separate them based on the size as they migrate towards the positive electrode

Protein Detection Methods

• After electrophoresis, the proteins can be visualized with a stain, such as Coomassie Brilliant Blue, that binds to protein non-specifically
• Gel transfer: transferring proteins from the gel to membranes such as the nitrocellulose using the electroblotting technique to make them accessible for binding the primary antibodies
• Electroblotting pulls out the negatively charged proteins using electric current

Protein Detection Steps

• Blocking: bovine serum albumin or milk solutions (contain proteins) are applied to the membrane to block or minimise non-specific binding
• Primary Antibodies, which are specific to the protein of interest, used to bind and target modified proteins (PTMs)
• Primary antibodies are linked to enzymes that generates colorimetric reactions when exposed to a substrate
• Rinsing: to remove unbound or weakly bound antibodies
• Secondary antibodies (conjugated to biotin or reporter enzyme such as the Horseradish peroxidase): binds the primary antibody to allow for detection and enhancing the signal of the protein of interest

SDS-PAGE Marker 1 2 3 4 5 6 7 8 1 - - 11 - -

Protein Expression and Purification in Research

• Fundamental steps in protein expression:
  1. Molecular cloning
  2. Cell culture - mostly E.coli
     1. SDS-PAGE to track if the protein is expressed or not
  3. Cell lysis - Lysis buffer > results in cell lysate (expressed proteins)
  4. Purification
     1. Protein binding to a matrix or resin
        1. The buffer is mobile phase
        2. The beads are the stationary phase
     2. Washing
     3. Elution

Protein Expression and Purification Techniques

• Protein expression in research is important to synthesize large quantities of the protein of interest
• Purification of these proteins is always conducted using high-performance liquid chromatography (HPLC)
• Common types of protein chromatography include;
  1. Size exclusion chromatography.
  2. Ion exchange HPLC.
  3. Affinity HPLC
  4. Reverse Phase HPLC

Introduction to Chromatography

Chromatography Terminology

Terminology	Definition
Mobile phase/carrier	Solvent moving through the column
Stationary phase/adsorbent	Substance that stays fixed inside the column
Eluent	Substance entering the column
Eluate	Fluid exiting the column (purified substance)
Elution	The process of washing out a compound through a column using a suitable solvent
Analyte	A mixture whose individual components have to be separated and analysed

High-Performance Liquid Chromatography (HPLC)

It is called high-performance liquid chromatography or occasionally high-pressure liquid chromatography Chromatography basically refers back to various types of separation methods These separation methods generally involve dissolving the sample of interest (analyte) into a mobile phase and forcing it via a particular type of stationary phase

HPLC Process

The mobile phase forced under high pressure through a chromatography column which is narrow and packed with a stationary phase Traveling of the mobile phase along the chromatography column results in separating the sample mixture into fractions which then collected from the column A constant addition of mobile phase will force the mixture to be separated and this technique called elution

Thin Layer Chromatography (TLC)

Definition: TLC is a very simple method easy to implement that was first developed in the 1940s Used today in the clinical laboratory mostly as a screening or semi-quantitative test for amino acids, proteins, drugs, and lipids in serum or urine

Size Exclusion Chromatography

This stationary phase separates a mixture of proteins using porous beads. Larger molecules flow faster than smaller molecules which stack between the beads. A wide range of pore sizes are available in the market for the separation of different types of mixtures.

Mixture of large and small proteins Size exclusion chromatography Chromatography column Tubes for collecting proteins after separation Eluate #1 Eluate #2

Ion Exchange Chromatography

This stationary phase of the HPLC is composed of charged functional molecules which pack the chromatography column. These charged molecules will bind to the opposite charged particles in the sample and the residual molecules will pass through the column. The elution process of the stack protein in the column involves changing the mobile phase's pH.

+ I + + + + + + Ion exchange chromatography

Affinity Chromatography

Involves applying particular molecules in the stationary phase which can target a specific protein of interest in the sample For instance, using an antibody which will bind specifically to that particular protein, while the rest of proteins in the sample will flow out the column Following that changing the mobile phase is needed to unbound the targeted protein from the antibody and elute it