session iii how we analyzed proteomic data? 台大生技教改暑期課程
TRANSCRIPT
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Session III Session III
How we analyzed proteomic How we analyzed proteomic data?data?
台大生技教改暑期課程
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Topics Topics for session IIIfor session III
1.1. Image analysis (for 2-DE gel)Image analysis (for 2-DE gel)
2.2. Mass data analysisMass data analysis
3.3. Protein structure analysisProtein structure analysis
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1. Image analysis1. Image analysis
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Examples of 2-DE resultsExamples of 2-DE results
D
Healthy control Patient
Digest to peptide fragment MS analysis
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Unexpected variation between gels Unexpected variation between gels
Interior variationInterior variation between 2-DE experiments between 2-DE experiments
– Same loading amount?Same loading amount?
– Same gel condition?Same gel condition?
– Same staining condition?Same staining condition?
Exterior variationExterior variation after gel developed after gel developed
– Unwanted spots (dye or reagent deposit)Unwanted spots (dye or reagent deposit)
– Dirty spots (hair, dust)Dirty spots (hair, dust)
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What image analysis software do? What image analysis software do?
– spot detectionspot detection
– unwanted spot filteringunwanted spot filtering
– background subtractionbackground subtraction
– normalizationnormalization
– image matchingimage matching
– expression comparisonexpression comparison
– pI/MW calibration pI/MW calibration
– data organizationdata organization
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Currently available Currently available 2-DE image analysis software 2-DE image analysis software
– Melanie 4 Melanie 4 (Swiss Institute of Bioinformatics, SIB)(Swiss Institute of Bioinformatics, SIB)
– Phoretix 2D Phoretix 2D (Nonlinear dynamics)(Nonlinear dynamics)
– Progenesis Progenesis (Nonlinear dynamics)(Nonlinear dynamics)
– Z3 and Z4000 Z3 and Z4000 (Compugen)(Compugen)
– Delta2D Delta2D (Sunergia group)(Sunergia group)
– A-GelFox 2D A-GelFox 2D (Alpha innotech)(Alpha innotech)
– Flicker Flicker (NCI, through internet)(NCI, through internet)
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Spot detection Spot detection
One of the first and most important steps in One of the first and most important steps in 2-DE analysis. 2-DE analysis.
Locating the spots in the gel imageLocating the spots in the gel image defining their shape defining their shape calculating measurement information calculating measurement information (volume and area)(volume and area)
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Spot detection Spot detection
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Filtering Filtering
Removal of unwanted spots:Removal of unwanted spots:
What’s unwanted spots: What’s unwanted spots:
dust on geldust on gel stain depositstain deposit bulky spotsbulky spots
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Background subtraction Background subtraction
General background subtraction method:General background subtraction method:
No backgroundNo background Mode of non-spotMode of non-spot Manual backgroundManual background Lowest boundaryLowest boundary Average boundaryAverage boundary
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Normalization Normalization
General normalization method:General normalization method:
Total spot volumeTotal spot volume Single spotSingle spot Total volume ratioTotal volume ratio
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Image matching Image matching
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Expression comparison Expression comparison
Two foldTwo fold up or down expression are thought up or down expression are thought to be significant.to be significant.
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pI/MW calibration pI/MW calibration
PI calibration MW calibration
observed or experimental pI/MWobserved or experimental pI/MW
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Data organization Data organization
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Spot annotation Spot annotation
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2. Mass data analysis2. Mass data analysis
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Useful proteomic resourceUseful proteomic resource
http://tw.expasy.org/
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Useful proteomic resourceUseful proteomic resource
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Matrix Science - MascotMatrix Science - Mascot
http://www.matrixscience.com/
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Three major functions in MascotThree major functions in Mascot
Peptide Mass Fingerprint (PMF): The experimental data are a list of peptide mass values from an enzymatic digest of a protein. (MALDI-TOF)
Sequence Query: One or more peptide mass values associated with information such as partial or ambiguous sequence strings, amino acid composition information, MS/MS fragment ion masses, etc. A super-set of a sequence tag query.
MS/MS Ion Search: Identification based on raw MS/MS data from one or more peptides. (LC/MS/MS)
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Difference between MALDI-TOF and LC/MS/MSDifference between MALDI-TOF and LC/MS/MS
MALDI-TOFMALDI-TOF
LC/MS/MSLC/MS/MS
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2-1 PMF analysis2-1 PMF analysis
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Raw data for PMF
899.2076 2980.8123905.2126 1471.3723909.1917 2312.2317915.2181 1533.8486925.4637 1881.7635938.3972 1528.94621044.3007 2111.94821050.3141 2396.15501060.2797 4689.06981066.2889 7302.00291072.2991 5688.85111078.3169 8919.11131084.2657 1474.59001088.2793 3180.51221094.2947 4573.51951104.2638 1546.46521110.2837 1470.97341271.3163 1498.0187
m/zm/z Relative intensityRelative intensity
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Mascot PMF query form
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Mascot PMF parameters
Your name; Email Search title Database Taxonomy Enzyme Monoisotopic or Average Modifications Protein Mass Peptide tol. ± Mass values Missed cleavages Data file Query
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Database
Database Comment
ESTEST divisions of Genbank,(currently EST_human, EST_mouse, EST_others)
MSDB Comprehensive, non-identical protein database
NCBInr Comprehensive, non-identical protein database
OWL Non-identical protein database (obsolete)
Random Random sequences for verifying scoring statistics
SwissProt High quality, curated protein database
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Taxonomy
Ensure the hit list will only contain entries from the selected species
speed up a search bring a weak match
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EnzymeName Cleave Don't cleave N or C term
Trypsin KR P CTERM
Arg-C R P CTERM
Asp-N BD NTERM
Asp-N_ambic DE NTERM
Chymotrypsin FYWL P CTERM
CNBr M CTERM
Formic_acid D CTERM
Lys-C K P CTERM
Lys-C/P K CTERM
PepsinA FL CTERM
Tryp-CNBr KRM P CTERM
TrypChymo FYWLKR P CTERM
Trypsin/P KR CTERM
V8-DE BDEZ P CTERM
V8-E EZ P CTERM
CNBr+TrypsinM CTERM
KR P CTERM
None see notes
semiTrypsin see notes
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Enzyme
"None" is not an allowed choice for a Peptide Mass Fingerprint, where the specificity of an enzyme is essential.
If the search fails to produce a positive match, then try again with semiTrypsin (below) before resorting to "None".
"semiTrypsin" means that Mascot will search for peptides that show tryptic specificity (KR not P) at one terminus, but where the other terminus may be a non-tryptic cleavage. This is a half-way house between choosing "Trypsin" and "None".
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Monoisotopic or Average nominal mass values:nominal mass values: calculated from integer atomic weights. (H=1, C=12, N=14, O=16), not practical in proteomics. Average mass values:Average mass values: equivalent to taking the centroid of the complete isotopic envelope Monoisotopic mass value:Monoisotopic mass value: the mass of the first peak of the isotope distribution.
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Monoisotopic or Average
For peptides and proteins, the difference between an average and a monoisotopic weight is approximately 0.06%.
Insulin (5.8 kD) Albumin (66.4 kD)
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Monoisotopic
Tol: 1 Da
-1.01
Monoisotopic MW
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Average
Tol: 1 Da
Average MW
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Monoisotopic
Tol: 2 Da
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Average
Tol: 2 Da
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Modifications Most protein samples exhibit some degree of modification. Natural post-translational modifications:Natural post-translational modifications: phosphorylation and glycosylation. Deliberate modifications:Deliberate modifications: deliberately introduced during sample work-up, such as cysteine derivatisation.
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Modifications
Fixed modificationsFixed modifications are applied universally, to every instance of the specified residue(s) or terminus.
Example: Carboxymethyl (Cys) means that all calculations will use 161 Da as the mass of cysteine.
Variable modificationsVariable modifications are those which may or may not be present.
Example: if Oxidation (Met) is selected, and a peptide contains 3 methionines, Mascot will test for a match with the experimental data for that peptide containing 0, 1, 2, or 3 oxidised methionine residues.
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Modifications
Fixed modificationsFixed modifications are applied universally, to every instance of the specified residue(s) or terminus.
Example: Carboxymethyl (Cys) means that all calculations will use 161 Da as the mass of cysteine.
Variable modificationsVariable modifications are those which may or may not be present.
Example: if Oxidation (Met) is selected, and a peptide contains 3 methionines, Mascot will test for a match with the experimental data for that peptide containing 0, 1, 2, or 3 oxidised methionine residues.
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Peptide tol. ±
% fraction expressed as a percentage
mmu absolute milli-mass units, i.e. units of .001 Da
ppm fraction expressed as parts per million
Da absolute units of Da
The error window on experimental peptide mass values
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Missed cleavages
Missed cleavage = 0, complete digestionMissed cleavage >=1, incomplete digestion
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Submit and processing
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Concise protein summary
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protein summary
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PMF protein view (I)
Score and ExpectProtein name
MW and pI
coverage
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PMF protein view (II)
Match peptides
No match peptidesRMS error
Protein information
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2-2 MS/MS analysis2-2 MS/MS analysis
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Raw data for MS/MS
Parent ion
Daughter ion
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Mascot MS/MS query form
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Protein summary
Most possible candidate
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MS/MS Protein view (I)
The sum of all highest scores within each peptide group
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MS/MS Protein view (II)Protein score: The sum of all highest scores within each peptide group
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Peptide view
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3. Protein structure analysis3. Protein structure analysis
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Research Collaboratory for Structur
al Bioinformatics (RCSB)
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Protein data bank (PDB)
Proteosome
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Example: 3D structure for proteosome
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Example: 3D structure for proteosome
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Example: 3D structure for proteosome
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Example: 3D structure for proteosome
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Stereo view
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Rotation
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Secondary Structure