Transcript Proteomics1_2012

Goals in Proteomics
1.
Identify and quantify proteins in complex mixtures/complexes
2.
Identify global protein-protein interactions
3.
Define protein localizations within cells
4.
Measure and characterize post-translational modifications
5.
Measure and characterize activity (e.g. substrate specificity, etc)
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Goals in Proteomics
1.
Identify and quantify proteins in complex mixtures/complexes
MS and MS/MS
2.
Identify global protein-protein interactions
MS and MS/MS, Y2H
3.
Define protein localizations within cells
High-throughput microscopy, organelle pull down
4.
Measure and characterize post-translational modifications
MS techniques
5.
Measure and characterize activity (e.g. substrate specificity, etc)
Protein arrays
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Basic overview of Tandem mass-spectrometry (MS/MS)
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Coon et al. 2005
Intro to Mass Spec (MS)
Separate and identify peptide fragments by their Mass and Charge (m/z ratio)
Mass Spec
Ion source
Mass analyzer
MS Spectrum
Detector
Basic principles:
1. Ionize (i.e. charge) peptide fragments
2. Separate ions by mass/charge (m/z) ratio
3. Detect ions of different m/z ratio
4. Compare to database of predicted m/z fragments for each genome
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Intro to Mass Spec (MS)
Separate and identify peptide fragments by their Mass and Charge (m/z ratio)
1.
Ionization
Goal: ionize (i.e. charge) peptide fragments without destroying molecule
Positive ionization (protonate amine groups)
especially useful for trypsinized proteins (cleaved after R and K)
vs.
Negative ionization (deprotonate carboxylics and alcohols)
http://www.colorado.edu/chemistry/chem5181/MS_ESI_Gilman_Mashburn.pdf
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Liquid chromatography + Electrospray ionization
electric field
* Commonly used with liquid solutions, more sensitive to contaminants, used for complex mixtures
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Liquid chromatography + Electrospray ionization
electric field
* Commonly used with liquid solutions, more sensitive to contaminants, used for complex mixtures
MALDI
* Less sensitive to contaminants, more common for less complex mixtures
http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
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Intro to Mass Spec (MS)
Separate and identify peptide fragments by their Mass and Charge (m/z ratio)
1.
Ionization
Goal: ionize (i.e. charge) peptide fragments without destroying molecule
2.
Separation of ions based on m/z ratio (mass m versus charge c)
Multiple flavors of mass analyzers use different technology.
Different mass analyzers can be hooked together for unique properties.
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Multiple flavors of mass analyzers
Single MS (peptide fingerprinting):
Identifies m/z of peptide only
Peptide id’d by comparison to database,
of predicted m/z of trypsinized proteins
Tandem MS/MS (peptide sequencing):
Pulls each peptide from the first MS
Breaks up peptide bond
Identifies each fragment based on m/z
Collision cell
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Multiple flavors of mass analyzers … can be hooked together in multiple configs.
g. Orbitrap
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Multiple flavors of mass analyzers
Single MS (peptide fingerprinting):
Identifies m/z of peptide only
Peptide id’d by comparison to database,
of predicted m/z of trypsinized proteins
Tandem MS/MS (peptide sequencing):
Pulls each peptide from the first MS
Breaks up peptide bond
Identifies each fragment based on m/z
Collision cell
Now multiple types of collision cells:
CID: collision induced dissociation
ETD: electron transfer dissociation
HCD: high-energy collision dissociation
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Fragmentation happens in fairly defined way along peptide backbone
Peptide can fragment along 3 possible bonds …
charge stays on either the Nt (a,b, or c) or Ct (x, y, or z) side of cleavagee
With CID, cleavage along the CO-NH bond is most common, generating ‘b’ and ‘y’ ions
* But phospho-modifications are labile via CID, and thus often missed
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Electron-transfer dissociation (ETD) targets different bonds
ETD cleavages favors ‘c’ and ‘z’ ions, is less sensitive to peptide sequence or length,
and does not target phospho bonds
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MS spectrum (i.e. peptide ions)
Each peak often surrounded
by smaller peaks of similar m/z
Sensitivity of instrument
determines resolution
Each peak is a different peptide, separated based on m/z
A single peptide is selected by the instrument for the second MS
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Mann Nat Reviews MBC. 5:699:711
Second MS identifies y (or b) ions to read out amino-acid sequence
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Mann Nat Reviews MBC. 5:699:711
Trypsin often used to digest proteins (cleaves after Arg and Lys)
Because of challenges distinguishing spectra, simplified mixtures
are typically injected into the MS:
-
-
either excised proteins
- purified complexes
fractionated pools of complex mixtures
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2 dimensional gel separation
(largely outdated)
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
(pH gradient makes the net charge 0)
The second dimension
(separation by mass)
-pH gel strip is loaded onto a SDS gel
-SDS denatures the protein (to make
movement solely dependent on mass,
not shape) and eliminates charge.
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Ahna Skop
TAP-tag: Tandem Affinity Purification
(for IP’ing individual proteins and proteins
bound to them)
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Ion exchange chromatography
Anion exchange:
Column is postively charged (can
bind negatively charged proteins).
Cation exchange:
Column is negatively charged (can
bind positively charged proteins).
Exploit the isoelectric point of a protein to
Separate it from other macromolecules.
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Ahna Skop
Affinity chromatography
A ligand with high affinity to the protein
is attached to a matrix.
Protein of interest binds to ligand
And is retained by resin. Everything else
flows through.
Can use excess of the soluble ligand
to elute the protein.
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Ahna Skop
Phospho-proteomics and Post-translational modifications (PTMs)
Phosphorylated (P’d) peptides are enriched, typically through chromatography
- P’d peptides do not ionize as well as unP’d peptides
- enrichment of P’d peptides ensures ionization and aids in mapping
IMAC: immobilized metal ion affinity chromatography
- phospho groups bind charged metals
- contamination by negatively-charged peptides
Titanium dioxide (TiO2) column:
- binds phospho groups (mono-P’d better than multi-P’d)
SIMAC: Sequential Elution from IMAC:
- IMAC followed by TiO2 column
Goal: identify which residues are phosphorylated (Ser, Thr, Tyr),
mapped based on known m/z of phospho group
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Quantitative proteomics
Either absolute measurements or relatively comparisons
1.
Spectral counting
2.
Isotope labeling (SILAC)
3.
Isobaric tagging (iTRAQ & TMT)
4.
SRM
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Spectral counting
counting the number of peptides and counts for each protein
Challenges:
- different peptides are more (or less) likely to be assayed
- analysis of complex mixtures often not saturating – may miss some
peptides in some runs
newer high-mass accuracy machines alleviate these challenges
- quantitation comes in comparing separate mass-spec runs … therefore
normalization is critical and can be confounded by error
- requires careful statistics to account for differences in:
quality of run, likelihood of observing each peptide, likelihood
of observing each protein (eg. based on length, solubility, etc)
Advantages / Challenges
+ label-free quantitation; cells can be grown in any medium
- requires careful statistics to quantify
- subject to run-to-run variation / error
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SILAC
(Stable Isotope Labeling with Amino acids in Cell culture)
Cells are grown separately in heavy (13C) or light (12C) amino acids (often K or R),
lysates are mixed, then analyzed in the same mass-spec run
Mass shift of one neutron allows deconvolution, and quantification,
of peaks in the same run.
Advantages / Challenges:
+ not affected by run-to-run variation
- need special media to incorporate heavy aa’s,
- can only compare (and quantify) few samples directly
- incomplete label incorporation can confound MS/MS identification
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Isobaric Tagging
iTRAQ or
Tandem Mass Tags, TMTs
Each peptide mix covalently tagged
with one of 4, 6, or 8 chemical
tags of identical mass
LTQ Velos
Orbitrap
Samples are then pooled and analyzed
in the same MS run
Collision before MS2 breaks tags –
Tags can be distinguished in the
small-mass range and quantified to
give relative abundance across
up to 8 samples.
Advantages / Challenges:
+ can analyze up to 8 samples,
same run
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- still need to deal with normalization
Selective Reaction Monitoring (SRM)
Targeted proteomics to quantify specific peptides with great accuracy
-
Specialized instrument capable of very sensitively measuring
the transition of precursor peptide and one peptide fragment
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Typically dope in heavy-labeled synthetic peptides of precisely known
abundance to quantify
Advantages:
- best precision measurements
Disadvantages:
- need to identify ‘proteotypic’ peptides for doping controls
- expensive to make many heavy peptides of precise abundance
- limited number of proteins that can be analyzed
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How does each spectrum translate to amino acid sequence?
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