Proteomics Guest Lecture Flashcards
What are the three main steps in high throughput proteomics?
- Sample preparation
- Data acquisition
- Data analysis bioinformatics
How were proteomic studies first carried out?
Two dimensional electrophoresis and amino acid analysis.
Samples were separated by their isoelectric piont AND size to sort proteins into groups.
You could also use mass spectrometry to identify bands on a gel. Subject them to digestion (usually by trypsin) to break them into peptides before mass spec analysis. Masses were determined with the spectrometry and these were matched with a database of peptide masses
How can you identify spots on a 2D proteomics gel?
- Amino acid analysis
- Isoelectric point
- Molecular weight
- Immunoblotting
- Sequencing
Able to look in database and come up with identity of proteins for a sample, no use of mass spectrometry.
Explain liquid chromatography mass spectrometry (LC-MS/MS)
Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS).
Give the steps of LC-MS/MS
What is different with tandem mass spec?
- A sample is digested, run on a gel and then injected into a liquid chromatography column where it is separated by hydrophobicity
- It is then injected into the mass spectrometer where it is ionized and sorted and then detected
There is an extra step between the ionization source and ion sorting in tandem mass spec (MS/MS). There is an analyzer and collision cell where the peptides collide with inert gas and fragment into inert pieces.
Describe the use of LC-MS in proteomics
LC-MS is used in proteomics where components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides.
LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer.
Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.
How is data from LC-MS/MS analyzed with bioinformatics?
- Compare empirical data with theoretical data from the database
- Search engines and algorithms compare your data to theoretical using the parameters that you used (organism, digestion enzyme used, which instrument etc.)
- Peptide validation and protein ID inferences are made
- Highest scoring spectra correpsonds to best peptide match. You can trace back through peptide fragment and back to protein in database.
What is shot-gun proteomics? How can this process be aided?
Where you skip the gel and instead just lyse cells and attack with trypsin to generate peptides which you can analyze in LC-MS/MS system.
This came about in the 90s when liquid chromatography and mass spec was improved.
This is the way it’s mostly done now.
This process can be aided by adding other steps of peptide fractionation (eg. MudPIT) before liquid chromatography to help reduce sample complexity (eg. centrifugation etc.)
True or false. The products of all genes in the genome are detectable by gel based and non-gel based proteomic approaches
False.
Some will only be detected by one or the other.
2D gel electrophoresis vs. 2D-LC
LC is gel free, which is less time but labour intensive! THere is a loss of protein MW and PTM information. These technologies are complementing, not alternative. Most studies use both.
How can LC-MS be used for quantitative studies of proteins?
You can label proteins of interest with a tag, one light and one heavy. You can then get a ratio of heavy to light (intensity).
Define proteomics
The study of the structure and function of proteins in a cell or tissue at a specific itme and under certain pre defined conditions; includes information on the way the proteins function and interact with each other inside cells.
Proteomics is no longer exclusive to 2D-gel electrophoresis.
