Lecture 4 Flashcards
How are intact proteins typically studied?
Studied by gel electrophoresis
What do the most common mass spectrometry workflows analyze?
The most common mass spectrometry workflows for complex protein samples analyze peptides, which are easier than proteins to fractionate by LC and ionize and fragment more efficiently than proteins, and the resulting spectra are easier to interpret for protein identification.
In Solution digestion strategies.
- For in-solution digestion strategies, proteins are denatured with strong chaotropic agents such as urea or thiourea. This step is either followed by or combined with disulfide reduction using a reducing agent such as dithiothreitol (DTT).
- The free sulfhydryl groups on the cysteine residues are then alkylated with reagents such as iodoacetamide to irreversibly prevent the free sulfhydryls from reforming disulfide bonds.
- The denatured, reduced and alkylated proteins are then digested by endoproteinases, (e.g., trypsin, chymotrypsin, Glu-C and Lys-C), which hydrolytically break peptide bonds to fragment proteins into peptides.
In-Gel Digestion strategies
- Protein separation by 1- or 2-dimensional gel electrophoresis is an alternative to in-solution protein denaturation, reduction, alkylation and digestion.
- It employs SDS polyacrylamide gel electrophoresis (SDS-PAGE) to denature and separate proteins in a sample.
- After electrophoresis, protein bands or spots are visualized using Coomassie, fluorescence or silver stains.
- Protein bands or spots are then excised from the gel and destained, and the proteins in the gel plugs are reduced, alkylated and digested in situ.
- The peptides are then extracted from the gel matrix and prepared for MS analysis.
What does the sample buffer often contain?
*Chaotropes, e.g. Urea and thiourea
*Ionic, non-ionic or zwitterionic detergent (e.g. SDS)
*Reducing Agent (e.g. DTT)
Chaotropes - Urea and Thiourea
- denaturing agent which solubilises and unfolds most proteins to their fully random conformation
- all ionisable groups are exposed to solution
- Urea often used at conc. of 5-7M, thiourea at 2M
- Competes with H-Bonds. Denatures proteins.
SDS
- SDS is an anionic detergent that disrupts non-covalent interactions in native proteins.
- ensures complete sample solubilisation and prevents aggregation through hydrophobic interactions
Reducing Agent
- e.g. DTT
- to break any disulphide bonds
- maintain proteins in their fully reduced state
- B-mercaptoethanol is an alternative
What is Physical Lysis
A common method of cell disruption and extraction of cellular contents. (e.g., dounce pestles, sonication, homogenisation).
Proteolysis
- enzymatic degradation of proteins into smaller peptides or amino acids by using site-specific endoproteinases (proteases).
- Small molecule measurements are more suitable for mass spec.
Trypsin
- “Gold Standard” for bottom up proteomics
- Digestive protease produced in pancreas.
- Trypsin cleaves at the carboxylic side of arginine and lysine residues.
- The sizes of the peptide fragments obtained after trypsin digestion, represent the peptide mass fingerprint and are characteristics of each protein.
- Protein sequences are stored in a database (i.e. UNIPROT)
- Sequences are digested in silico (based on the appropriate protease)
- Peptide masses are measured and MS/MS spectra recorded.
- Peptide sequence candidates which match the mass of the peptides measured (within a certain tolerance) are selected and a theoretical MS/MS spectrum is generated (i.e. a bar-code)
- Bar-code is then matched to the MS/MS spectrum.
What are challenges with Proteolysis?
What are the solutions used to combat these challenges?
Challenges:
- Protein folded too tightly - Protease can’t access
- Protein is insoluble and require additives.
Solutions:
Solutions:
* Urea, Guanidine HCl and organic solvents (e.g., acetonitrile) – denaturing agents
* Detergents such as Triton X-100 and SDS - these solubilize proteis
Limitations of Proteolysis
- Protease Inhibition
- Unwanted side effects (i.e., protein modification).
- Detergents hurt the hardware (LC and Mass Spec)!
Challenges with Trypsin
- Some tryptic peptides are too long or too short for Mass Spec analysis.
- Trypsin cleavage sites might be blocked due to post-translational modifications (PTMs) like phosphorylation or glycosylation.
- Certain proteins, such as membrane proteins or tightly folded proteins, are not efficiently digested by trypsin.
- Additional proteases allow for more sequence coverage and protein ID’s.
Alternative Proteases for Mass Spec
- Chymotrypsin
- Thermolysin
* More on slide 19
Chymotrpsin
- Serine endoproteinase derived from bovine pancreas.
- The protease preferentially hydrolyzes at the carboxyl side of aromatic amino acids: tyrosine, phenylalanine and tryptophan.
- Chymotrypsin activity is optimal at pH 7.0–9.0
Thermolysin
- A thermostable metalloproteinase.
- Thermolysin preferentially cleaves at the N-terminus of the hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine and methionine.
- The optimal digestion temperature range is 65–85°C. Thermolysin activity is optimal at pH 5.0–8.5.
What is the impact of adding alternative proteases?
- Adding additional proteases improves proteomic analysis
HIgh Performance Liquid Chromatography - what does it involve?
- It is a seperation technique that involves.
- The injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)
- where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.
- Components seperated by coluimn packing
- Seperated components are detect at the column by a dector that measures their amount
* Slides 25-26
What are the four major seperation modes that are used to seperate most compounds?
1.Reversed-phase chromatography 2.Normal-phase and adsorption chromatography
3.Ion exchange chromatography 4.Size exclusion chromatography
Slide 29- 33 Diagrams and info on reverse phase chromatography
What is seperation based upon?
Based upon differential migration between the stationary and mobile phases
Stationary Phase
the phase which remains fixed in the column,. e.g C18, Silica.
Mobile Phase
Carries the sample through the stationary phase as it moves through the column.
