Shotgun proteomics
“The shotgun proteomic strategy based on digesting proteins into peptides
and sequencing them using tandem mass spectrometry.”
Definition:
Shotgun proteomics is a method of identifying proteins in complex mixtures using liquid chromatography combined with mass spectrometry. The name is derived from shotgun sequencing of DNA.
Around the globe:
The regions which are mainly involved in shotgun proteomics research are;
- USA (California, Colorado, Washington, Boston, Tennessee, etc)
- Singapore
- China (Harbin)
History:
Regarding the origin of this technique, no specific name is found in history. However the evolution comes from the work of various scientists during last decade.
Shotgun Proteomics Technique:
Firstly, the proteins from the mixture are digested. Then, the resulting peptides are separated liquid chromatography. Then they are identifies by Tandom mass spectrometry.
Digestion of Proteins:
Proteins are broken down into peptides by treating them with different enzymes;
· amylase – breaks down carbohydrates, starches, and sugars
· protease (trypsin, pepsin, etc) – breaks down proteins found in meats, nuts, eggs, and cheese
· lipase – breaks down fats found in most dairy products, nuts, oils, and meat
· cellulase – breaks down cellulose, plant fiber
Separation of peptides:
Typical techniques used for separation of pepetides are; 2D PAGE, HPLC, CE, DC, etc. In Shotgun Proteomics, HPLC is used.
HPLC:
It is a chromatographic technique that can separate a mixture of compounds. High-performance liquid chromatography or high-pressure liquid chromatography.
HPLC is highly improved form of basic chromatography. In it the solvent is forced through under high pressures of up to 400 atmospheres to drip through a column, instead of being allowed to drip under gravity. This makes it much faster. It also allows a much better separation of the components of the mixture.
Forms of HPLC:
Depending on the relative polarity of the solvent and the stationary phase, there are two variants used in HPLC.
- Normal phase HPLC
- Reversed phase HPLC
Normal phase HPLC:
It is not the most commonly used form of HPLC. The column is filled with tiny silica particles, and the solvent is non-polar (for example: hexane). A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column.
Reversed phase HPLC:
In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them. A polar solvent is used (for example: a mixture of water and an alcohol such as methanol).
There will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds. Hence, they spend less time in solution in the solvent and this will slow them down on their way through the column.
Therefore, the polar molecules will travel through the column more quickly. Reversed phase HPLC is the most commonly used form of HPLC.
Retention time:
The time taken for a particular compound to travel through the column to the detector is known as its retention time. This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.
The Detector:
There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption.
Many organic compounds absorb UV light of various wavelengths. If we have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, we can get a direct reading of how much of the light is absorbed.
The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.
Interpreting the output from the detector:
The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector and absorbing UV light.We can use the peaks as a way of measuring the quantities of the compounds present.
Let's suppose that we are interested in a particular compound, X. If we injected a solution containing a known amount of pure X into the machine, not only could we record its retention time, but we could also relate the amount of X to the peak that was formed.
The area under the peak is proportional to the amount of X which has passed the detector, and this area can be calculated automatically by the computer linked to the display.
If the solution of X was less concentrated, the area under the peak would be less - although the retention time will still be the same.
This means that it is possible to calibrate the machine so that it can be used to find how much of a substance is present - even in very small quantities.
Advantages of HPLC:
- Easier automation (no gel handling)
- Better separation power (Multi-dimensional chromatography)
- Simpler extension to MS
Disadvantages of HPLC:
- Proteins are chopped up
- Quantitation difficult
· Data hard to manage / interpret
PatternLab – Software on Shotgun Proteomics:
PatternLab is a “simple to use” yet efficient software to addresses shotgun proteomic data analysis.
It was initialy published in BMC Bioinformatics in 2008. PatternLab has continued to evolve; an updated manuscript is found in Current Protocols in Bioinformatics.
This project was development by joint efforts from the Systems Engineering and Computer Science Program at the Federal University of Rio de Janreiro (Brazil) and the Biological Mass Spectrometry Laboratory (Yates Lab) from the Scripps Research institute (La Jolla, California).
Here are a few of the PatternLab most used modules;
- The ACFold and Tfold
- Points differentially expressed proteins in LC-MS experiments.
- The Gene Ontology Explorer (GOEx)
- Aids in the biological interpretation of shotgun proteomic data.
- It has an effective GUI.
- It stands out for providing data such as the global protein fold changes for the GO groups.
- The Charge Prediction Machine (CPM)
- Predicts the charge states of precursor ions of low resolution.
- Enable confident protein identification with low resolution equipment.
- YADA
- Can deisotope and decharge highly charged peptides.
- Assign monoisotopic precursor masses to MS.
- XDIA
- Introduces a new strategy for data acquisition in the mass spectrometer that relies on multiplexed spectra.
- The XDIA processor is required for protein identification.
Shotgun proteome Analysis
– Based on Tandem mass spectrometry
– Based on biomarker candidates
– Application of these techniques in Cancer proteomics
– Research paper-example
– Future of Shotgun proteomics
– Software applications
Shotgun proteome analysis based on liquid chromatography- tandem mass spectrometry:
This is a very popular approach in proteomics called "shotgun LC-MS/MS" strategy. In this technique,total protein digest is separated by ion exchange fractionation in the first dimension followed by off- or on-line RP LC-MS/MS. The peptides are separated by their isoelectric point in the first dimension and hydrophobicity in the second dimension
Shotgun proteomics to discover biomarker candidates in tissue specimens:
• The tissue or biofluid specimens are digested to tryptic peptides and then fractionated by isoelectric focusing (IEF) ;peptides in each IEF fraction are then analyzed by reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). MS spectra is generated from peptides in the sample and each MS/MS spectrum encodes the sequence of a peptide ;these sequences are determined by searching the spectra against a database of sequences corresponding to all known human proteins. The identified peptide sequences are then assembled into an inventory of proteins and the numbers of spectra that map to each protein provide a preliminary estimate of the amount of each protein in the sample.The Comparisons of the MS/MS datasets for tissue specimens (e.g., normal vs. cancer) thus allows the identification of proteotypes, which are the features of proteomes that distinguish one tissue type from another.
Research article: Shotgun proteome analysis of hibernating Arctic ground squirrels(Shao C.et al)
Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection.The previous gene expression studies of hibernation have mainly focused on changes at the mRNA level but large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species.so scientists constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression. more than 3,000 unique proteins from livers of arctic ground squirrels were identified .Among them, 517 proteins showed significant differential expression comparing animals sampled.Proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed (in both late torpid and early aroused animals compared with non-hibernating animals) whereas proteins involved in fatty acid catabolism were significantly overexpressed. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals. In late torpid animals, no significant changes at the mRNA levels between these stages had been observed. Results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.
Software:
Transproteomic Pipeline(TPP) :
is a completely free software solution for MS/MS-based shotgun proteomics analysis.The modules include:
• Probability Assignment and Validation module(peptide prophet)
• Protein Quantification module(Xpress)
• GUI- (PETUNIA)
• Spectral Library Building and Searching
• ID curation (*.out files into a single HTML-SUMMARY file ready for use with INTERACT )
• Input Processing: Search-Engine to pepXML converters
• Sequest, Mascot, ProbID, X!Tandem, and Phenyx.
DB -PARSER:
DBParser is a web-based program for shot-gun proteomics that takes the flat file output from a Mascot Search, or series of Mascot searches, and stores data in a MySQL database.
References:
8. http://onlinelibrary.wiley.com/doi/10.1002/pmic.v10:1/issuetoc
9. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B8JDC-50VCWC3-1&_user=10&_coverDate=10%2F10%2F2010&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ce20ea657c3db5281179483106936869&searchtype=a
10. Shao C, Liu Y, Ruan H, Li Y, Wang H, Kohl F, Goropashnaya AV, Fedorov VB, Zeng R, Barnes BM, Yan J. Shotgun proteomics analysis of hibernating arctic ground squirrels.Chinese Academy of Sciences-German Max Planck Society(CAS-MPG) Partner Institute for Computational Biology, Shanghai Institutes of Biological Sciences,320 Yue Yang Road, Shanghai 200031, China. Mol Cell Proteomics. 2010 Feb;9(2):313-26. Epub 2009 Nov 20.
11. www.genebio.com/products/phenyx/
12. http: //tools.proteomecenter.org/TPP.php
13. http://tools.proteomecenter.org/wiki/index.php?title=Main_Page
14. www.ncbi.nlm.nih.gov/pubmed/15473689
15. http://pubs.acs.org/doi/abs/10.1021/pr049920x
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