mass spectrometry and proteomics - lecture 6 · advantages and disadvantages of ge-lc advantages:...
TRANSCRIPT
Previously
• Quantitation techniques– Label-free– TMT– SILAC
• Peptide identification and FDR
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Lecture 6
• Chromatography• Fractionation• PTMs• Proteomics experiments
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Reversed phase chromatography
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www.lamondlab.com
Liquid chromatography
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Liquid chromatography
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Why fractionate?
1. Complexity of proteomes – 15,000 genes transcribed, ~100,000 isoforms, millions of post-translationally modified protein species.
2. Protein concentration range at least 106 in cell, 109 in serum.
3. Linear dynamic range of mass spectrometer ~103.
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Orthogonality
Non-orthogonal Orthogonal
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Common Protein and PeptideFractionation techniques
Protein Fractionation• Gel electrophoresis (GE) LC-MS• Macroporous Reversed Phase (MRP)
Peptide Fractionation• Reversed-phase liquid chromatography (RP LC)• Strong Cation Exchange Chromatography (SCX)• Isoelectric focusing (IEF)
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GE LC-MS
• In-gel digestion
• peptide extraction
OrbiTrapMS/MS
LC
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Advantages and Disadvantages of GE-LC
Advantages:• Simple and powerful approach due to high resolution.• High protein identification rate as not all peptide leave
the gel decomplexification.• Proteins are distributed over several bands (identification
of specific isoforms).
Disadvantages:• Not all peptide can be extracted low sequence
coverage (can be an advantage too!).• Only trypsin works well as an in-gel protease.• Proteins are distributed over several bands
(quantification difficult).
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Liquid Chromatography
HPLC
• Separation of different molecules by different adsorption to the stationary phase.
• Most common: reversed-phase high performance liquid chromatography (RP HPLC) using an aqueous mobile phase and non-polar/hydrophobic stationary phase such as C18.
• Ion exchange chromatography (ISCX): Separation of molecules depending on their charge
waters.com
Ion exchange chromatography
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www.waters.com
SCX SAX• Allows the separation
of peptides or proteins by charge.
• Charge of peptides and proteins is pH-dependent and thus, pH is important.
• Peptides are eluted by either replacement with salts or by a pH gradient. At their isoelectric point, peptides/proteins have no net charge and elute.
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SCX-Chromatography
• positively charged peptides (or proteins) bind to negatively charged stationary phase.
• in a 2D-LC-MS experiment peptides of each protein are spread over practically all fractions.
Strong cation exchange material
SAX chromatography
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• Uses mostly quaternary ammonium ions.• Advantages:
• many peptides Glu or Asp residues. • highly orthogonal to RP.• Can also be used to remove non-ionic
detergents.• pH or salt gradient.
Fractionation by SAX is highly orthogonal
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Ritorto et al, JPR, 2013
High-pH Reversed Phase• Changing the pH from acidic (pH<3) to basic (pH=10)
changes retention times of peptides on C18 reversed phase columns.
• Makes use of the high resolution of RP-LC.• Not orthogonal to low-pH RP LC. Can be avoided by
fraction concatenation (e.g. mixing fraction 1+11+21, 2+12+22 etc).
• Requires removal of organic solvents, thus not useful for online 2D.
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Hydrophilic interaction liquid chromatography (HILIC)
• HILIC is a variant of normal-phase liquid chromatography.
• The stationary phase is polar, but nor ionic. Often silanol or diol phases are used.
• Analytes are eluted by a gradient from high organic (mostly ACN) to high H2O.
• When an ionic stationary phase is used, the chromatography is called electrostatic repulsion hydrophilic interaction chromatography (ERLIC).
• Both separations have been successfully used, particularly in phosphoproteomics.
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Size Exclusion Chromatography (SEC)
• Can separate intact protein complexes from cell lysates.
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Protein correlation profiling (PCP) by Size Exclusion
Chromatography (SEC)
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• More instrument time required
• Extensive bioinformatics required
Workflow SEC
M. Larance
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Cross-Validation of SEC Profiles
Kirkwood & Larance et al, MCP 2013
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Analysis of post-translational modifications by MS
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Mann & Jensen 2003
Also important:• Oxidation (M, C, Y)• Phosphorylation
(H, R)
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Quantitative Phosphoproteomics
• Phosphorylation is a common post-translational protein modification.
• O-phosphorylation on Ser/Thr/Tyr, • N-Phosphorylation on Arg/Lys/His• Estimated 1,000,000 phosphorylation sites in
human.• One third of proteins are estimated to be
phosphorylated.• Protein phosphorylation regulates virtually all
aspects of the cell.• ~100,000 phosphorylation sites identified.
Trost et al, Mass Spec Reviews, 2010
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Phosphopeptide enrichment methods
Due to suppression effects and low abundance, phosphopeptides usually need to be enriched before MS analysis.• Strong cation (SCX) or strong anion exchange chromatography (SAX)• Immunoaffinity Chromatography (mostly pTyr-antibody)• Immobilized Metal ion Affinity Chromatography (IMAC)• Metal Oxide Affinity Chromatography (MOAC)• Chemical enrichment
Fragmentation of phosphopeptides
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m/z 1105.56502+ Mascot score 54 R.TLSDYNIQKESTLHLVLR.L + P
Fragmentation of phosphopeptides
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Chemical Enrichment• only target Ser/Thr-residues• problems through side reactions• so far, rarely used.
Trost et al, Mass Spec Rev, 2010
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Affinity enrichment• common IMAC metal
ions include Fe3+, Ga3+, Al3+, Zr4+
• IMAC and TiO2 seem to bind different phospho-peptide populations.
• TiO2 has the advantage of being inert and therefore virtually all buffers can be used.
• phosphopeptide enrichment by TiO2 is enhanced by addition of certain carbonic acids such as lactic acid of dihydrobenzoic acid (DHB)
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Absolute Quantitation of phosphopeptides
Trost et al, Mass Spec Rev, 2010
Algorithms to determine phosphosite probabilities
• A-Score (Gygi lab) – Sequest• Mascot Delta Score (Küster lab) - Mascot• PhosphoRS (Mechtler lab) – Proteome Discoverer• LuciPHOr (Nesvizhskii lab) – TPP• PTM Score (Mann lab) - MaxQuant
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Ubiquitin proteomics
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Quantitative pull-downs
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Quantitative pull-downs
Things to consider:1. Overexpressed, tagged or endogenous?2. SILAC?3. Do I use the best model?4. How much can be pulled down?5. Co-IP or PTMs? washes6. Be careful with detergents!
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Total cell proteomics
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Total cell proteomics
Things to consider:1. How deep do you need to go?2. What is more important? Accuracy or depth?3. Label-free, SILAC or TMT?
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Targeted Proteomics
229https://www.pmiscience.com/
Selected Reaction Monitoring (SRM)
Parallel Reaction Monitoring (PRM)
Targeted & absolute quantitation
230http://www.origene.com/
QConCat
231Pratt et al, Nature Protocols, 2006
Targeted & absolute quantitation
Things to consider:1. Is your target really the best target?2. Can you enrich it/pull it down?3. Do I measure it in the right sample?4. How accurate do you need it?5. Do I need absolute values?
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SWATH - Data Independent Acquisition (DIA)
233Imsb.ethz.ch
LOPIT
234Mulvey et al Nature Protocols, 2017
LOPIT
235Laurent Gatto
Questions?
• Matthias [email protected]
0191 2087009Cookson 4th floor
• [email protected]@ncl.ac.uk
0191 2084804
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