Session III Session III

How we analyzed proteomic How we analyzed proteomic data?data?

台大生技教改暑期課程

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

1. Image analysis1. Image analysis

Examples of 2-DE resultsExamples of 2-DE results

D

Healthy control Patient

Digest to peptide fragment MS analysis

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)

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

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)

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)

Spot detection Spot detection

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

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

Normalization Normalization

General normalization method:General normalization method:

Total spot volumeTotal spot volume Single spotSingle spot Total volume ratioTotal volume ratio

Image matching Image matching

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.

pI/MW calibration pI/MW calibration

PI calibration MW calibration

observed or experimental pI/MWobserved or experimental pI/MW

Data organization Data organization

Spot annotation Spot annotation

2. Mass data analysis2. Mass data analysis

Useful proteomic resourceUseful proteomic resource

http://tw.expasy.org/

Useful proteomic resourceUseful proteomic resource

Matrix Science - MascotMatrix Science - Mascot

http://www.matrixscience.com/

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)

Difference between MALDI-TOF and LC/MS/MSDifference between MALDI-TOF and LC/MS/MS

MALDI-TOFMALDI-TOF

LC/MS/MSLC/MS/MS

2-1 PMF analysis2-1 PMF analysis

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

Mascot PMF query form

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

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

Taxonomy

Ensure the hit list will only contain entries from the selected species

speed up a search bring a weak match

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

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".

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.

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)

Monoisotopic

Tol: 1 Da

-1.01

Monoisotopic MW

Average

Tol: 1 Da

Average MW

Monoisotopic

Tol: 2 Da

Average

Tol: 2 Da

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.

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.

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.

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

Missed cleavages

Missed cleavage = 0, complete digestionMissed cleavage >=1, incomplete digestion

Submit and processing

Concise protein summary

protein summary

PMF protein view (I)

Score and ExpectProtein name

MW and pI

coverage

PMF protein view (II)

Match peptides

No match peptidesRMS error

Protein information

2-2 MS/MS analysis2-2 MS/MS analysis

Raw data for MS/MS

Parent ion

Daughter ion

Mascot MS/MS query form

Protein summary

Most possible candidate

MS/MS Protein view (I)

The sum of all highest scores within each peptide group

MS/MS Protein view (II)Protein score: The sum of all highest scores within each peptide group

Peptide view

3. Protein structure analysis3. Protein structure analysis

Research Collaboratory for Structur

al Bioinformatics (RCSB)

Protein data bank (PDB)

Proteosome

Example: 3D structure for proteosome

Example: 3D structure for proteosome

Example: 3D structure for proteosome

Example: 3D structure for proteosome

Stereo view

Rotation

Secondary Structure