BIO312 Wang's part

Revision protein structure and methods of analysis

Techniques for protein analysis

Protein seperation

1. SDS-PAGE: basis-molecular weight
2. Isoelectric focusing: electric charge difference
3. Chromatic methods-useful adjuncts to gel-based approaches
   1. HPLC - high performance liquid chromatography
   2. Thin-layer chromatography
4. 2D gel electrophoresis
   • Powerful gel-based method commonly used to analyze complex samples
   • To characterize full range of proteins in the sample, not just a few specific proteins

SDS-PAGE:

• Proteins are usually mixed with the detergent, SDS and a tracking dye to make the sample
• SDS binds to the protein and gives it size-dependent negative charge and consistent hydrodynamic properties

Isoelectric point (pi)

• ph at which molecule has net zero charge （determined using computer program for known sequence or empirically ）

Isoelectric Focusing

Electrophoresis through polyacrylamide gel in which there is a pH gradient

2D gel

Strength and weakness of the 2D gel

Western Blot

a.k.a: immunoblotting

• Proteins first electrophoreses by SDS-PAGE
• Wet gel is placed against nitrocellulose
• In electrophoretic chamber:

Salting out (precipitation in protein fraction)

Ammonium sulfate precipitation is one of the most commonly used methods for protein purification from a solution.

In solution, proteins form hydrogen bonds with water molecules through their exposed polar and ionic groups. When high concentrations of small, highly charged ions such as ammonium sulfate are added, these groups compete with the proteins to bind to the water molecules.

This removes the water molecules from the protein and decreases its solubility, resulting in precipitation.

Critical factors that affect the concentration at which a particular protein will precipitate include: the number and position of polar groups, molecular weight of the protein, pH of the solution, and temperature at which the precipitation is performed.

The concentration at which antibodies precipitate varies among species; most rabbit antibodies precipitate with a 40% saturated solution, whereas mouse antibodies require 45-50% saturation.

Protocol

1. Allow serum or ascitic fluid to thaw, determine total volume, and centrifuge at 3000g for 30 minutes.
2. Transfer sample to a beaker containing a stir bar and place on a magnetic stirrer.
3. While the sample is stirring, slowly add saturated ammonium sulfate to bring the final concentration to 50% saturation.
   1. Volume of ammonium sulfate needed is equal to the volume of sample.
   2. Adding the ammonium sulfate very slowly ensures that local concentration around the site of addition does not exceed the desired salt concentration.
4. Once the total volume of ammonium sulfate is added, move beaker to 4°C for 6 hours or overnight.
5. Transfer to conical tube and centrifuge the precipitate at 3000g for 30 minutes.
6. Carefully remove and discard supernatant. Invert conical tube and drain well. For serum or ascites, resuspend pellet in 30%-50% of the starting volume in 1XPBS. For monoclonal antibody tissue culture supernatants, resuspend pellet in 10% of the starting volume in 1X PBS.
7. Transfer antibody solution to dialysis tubing and dialyze versus three changes of 1XPBS/0.08% Sodium Azide. Be sure to allow enough space for expansion of the antibody solution during dialysis. Normally twice the re-suspended volume is sufficient.
8. Remove antibody solution from the tubing and centrifuge to remove any remaining debris.
9. Determine the concentration and store at -80°C for long-term storage.

Centrifugation

chromatography

Gel filtration/Size exclusion chromatography

Ion exchange chromatography

Hydrophobic interaction chromatography

Protein sequencing

Sanger sequence (protein identification)

Cleavage of polypeptides for analysis

1. Strong acid - not sequence specific
2. Sequence-specific proteolytic enzymes (proteases)
3. Sequence-specific chemical cleavage (cyanogen bromide cleavage at methonine residues)

Only used in the mutant protein that is not in the databases

FPLC:

HPLC: high pressure

Protein identification

Edman degradation

Sequence aa in a peptide.

Amino-terminal residue is labeled and cleaved from the pipetted without disrupting the peptide bonds between other amino acid residues

Mass spectrometry

Mass Spectrometry:

Analytical techniques that measures the mass-to-charge ratio of charged particles, mass spectrometry is used for determining masses of particles and the elemental composition of a sample of molecules as well as for elucidating the chemical structure of molecules such as peptide

Workflow

Types of MS data

3D structure determination

• X-Ray Diffractionneed to crystalize (can be difficult)
  • 
• NMR small proteins (<25kD)
  • 
• Electron Micrographpoor resolution
  • 

Identification of proteins in complex mixtures

Proteomics achievement

Protein Identification by Mass Spectrometry

Mass Spec (MS) Basics

MALDI

Mass resolution

LC-MS/MS for protein identification

Mass spectrometer is always right

Advanced proteomic techniques

Summary of existing technology

ICAT

Top-Down or Bottom-up analysis

Top-down advantages

disadvantages for top-down

Quantitative Mass Spec-based Proteomics

Outline

• Principles of quantitative proteomics

• Gel vs. non-gel approaches

• Labeling vs. non-labeling approaches

• Case study: candidate biomarker generation using label-free approaches

Summary of label methods

Pros and Cons for label-free methods

Methods

Spectral counting & Peak-intensity

Spectral counting

Peptide Ion Intensity Based Protein Quantification

LC-MS Chromatographic Alignment

Limitations of LC-MS-based Approach to Large-scale Protein Profiling

Challenges in proteomics

Tutorial 1

Proteomics workflow

Separation technique

Mass spectrometry

Technique summary

PMF

TMS

• 注意这儿是Tandem MS sequence confirmation

The principle of SDS-PAGE

Western plot

Tutorial 2

Labeling vs non-labeling

comparison between label & label free

Label free approaches

pros and cons of label free protein

• Pros:

  • Technically simple
    • No complicated (or expensive) labelling or tagging protocol
  • Can avoid complex pre-separation
    • direct LC-MS is okay

• Cons:

  • Still can be expensive
    • Need plenty of replicates to get statistical power (machine time!)
  • Need good data analysis software
  • Not straightforward to validate results from big data sets
  • Low abundance proteins hard to measure accurately

Why does MS1 intensity not tell us peptide abundance directly?

• Peptide sequences ionise to different extents
  • based on the biophysical properties of a given sequence
• Thus, measured ion intensity only weakly correlates to the abundance of the peptide (and hence) protein in the proteome
• Not simple to extract the total ion abundance for a given peptide in different runs
• Difficult to ensure that you are comparing the same signal in parallel runs

Spectral counting

The normalization of spectral counts

Intensity based label free

Method summary:

• Compare MS1 peptide ion abundance across replicates runs;
• Specialist software must align different parallel LC-MS runs
• Calculate ratios from aligned MS1 data
• Requires good software (e.g.: Mascot distiller; Progenesis; MaxQuant; OpenMS; ProteomeDiscoverer; PLGS, many others…)

Advantages:

• In theory, should be more accurate than spectral counting – uses real intensity data
• No complicated labelling protocols

Disadvantages:

• Data processing is fairly CPU intensive
• Only works well if experimental system has high technical and biological reproducibility

Requirements:

• Very good LC delivery system (must be reproducible)
• High resolution mass spec (Orbitrap, Synapt etc…)
• Good PC for running software

Labelling quantification

Two approaches

1. Chemical (in vitro) labeling

   • ICAT (isotope coded affinity tag)
   • iTRAQ (isobaric tag for relative and absolute quantification)

2. Metabolic [or in vivo] labelling

   1. SILAC [stable isotope labelling by amino acids in culture]
      • Requires a cell culture, therefore cannot be used for experiments from other kinds of samples e.g. clinical samples

Lecture 5_Post-translational modifications

Proteomic Analysis for PTMs

Key issue in PTM analysis: modified proteins/peptides often have low abundance

• **Enrichment**can be used to increase their concentration in the sample

• The most established methodology is phosphoproteomics

• Development of phosphoproteomicshas been driven by the deep interest in signalling research (pharmaapplications)

• Phosphopeptides are relatively easy to enrich and mass spectrometric methods have been well established, however, the enrichment outcomes are often sample-dependent

• The field is currently moving to high-throughput of profiling methods of other PTMs and the analysis of several PTMs in parallel

MS for Phosphopeptides

• Precursor/ Reporter ion scanning
• Neutral-Loss-Dependent

Targeted Proteomics

Comparison between traditional and MS based protein quantification

MRM / SRM

Mass Spec-based Assay Benefits

Weakness of MRM/SRM