proteomics
DESCRIPTION
ProteomicsTRANSCRIPT
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ProteomicsProtein identification using mass spectrometry
www.draptis.eu/proteomics.ppt
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Introduction
Proteomics important technology Large-scale study of proteins
Structure Functions
“Omics” revolution: shift in strategy piece-by-piece -> global analysis hypothesis-driven -> discovery-based research
Expression proteomics analysis of differential protein expression
Functional proteomics posttranslational modifications protein-protein, protein-ligand interactions sequence-structure-function relationships
Twyman RM (2004). Principles Of Proteomics. Oxford, UK: BIOS
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Proteins Biochemical compounds
1 > polypeptides
Single linear polymer chain of amino acids (AA)
Bonded together by peptide ponds – carboxyl & AA residues
”Proteins." Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Amino acid sequence
”Proteins." Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Protein structure
Weaver, Molecular Biology, McGraw Hill: Boston, 2002
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Proteome
Entire set of proteins expressed by Genome Cell Tissue Organism
> 400.000 proteins, dynamic
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Human proteome
Aebersold et al, Systems biology - ETH Zurich, https://www.e-pics.ethz.ch/
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Why proteomics?
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Why proteomics?
Protein alterations cannot be fully deduced from DNA.
RNA expression does not always reflect protein levels: translational control degradation turnover
Some tissues not suitable for RNA expression analysis.
Proteins are the physiological/pathological active key players.
General goal: better understanding of genesis and progression of diseases
Clinical goals: early disease detection (biomarkers) identification of therapeutic targets therapy monitoring
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Multidisciplinary
TOF, Q, ITMALDI, ESIHPLCCells, tissue Algorithms
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Typical stages
Aebersold R, Mann M, Nature. 2003
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Quantification strategies
Domon B, Aebersold R. Science. 2006
Isoto
pe
dilu
tion
Tan
dem
mass t
ag
s
Inte
rnal
sta
nd
ard
s
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Top down or bottom up?
Bottom-up Most common Starting with
proteolytic fragments Piecing the protein
back together de novo repeat
detection
Top down Tandem MS of whole
protein ions Pulling them apart
Electron capture dissociation
Extensive sequence information
Fragment ions of
peptides
MS/MS
Proteolytic digeste.g. Trypsin
Protein
MS/MS
Fragment ions of protein
Bott
om
-up
Top d
ow
n
”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Separation Specific protein biophysical
parameters Isoelectric point Molecular weight Affinity
Chromatographic methods HPLC 2D-HPLC ProteinChips
Electrophoretic methods SDS-PAGE 2-D E
Reverse phase (RPLC) – Hydrophobicity
Yates JR, et al. Annu Rev Biomed Eng. 2009
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2D Gel electrophoresis
1D: isoelectric focussing (IEF) separation by IP
2D: dimension: SDS-PAGE separation by MW staining > 1000 proteins /gel
molecular analysis by MS HPLC Westernblot
Pitfalls very basic / acidic; large / small; hydrophobic; low-abundance proteins
Fontana et al. Proteomics 2004
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Robotic isolation
”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Shotgun proteomics
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HPLC
”HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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HPLC
HPLC/MS
Peptides from protein mixture fractionated in steps
Eluent ESI-MS MALDI: series
”HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Protein & peptide fractionation
Complex mixtures Proteins Molecules
Works only if mixture has equal amounts Abundant species suppress signals from less abundant ones Difficult to interpret Enyzmatic digestion -> many peptide products
2-D electrophoresis Protein fractionation
High performance liquid chromatography Peptide fractionation
”Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Proteins Peptides Fragments
Proteases, e.g. trypsin, break protein into peptides
Tandem MS breaks peptides into fragment ions Measures the mass of each piece. MS accelerates the fragmented ions; heavier ions accelerate slower than lighter ones MS measure mass/charge ratio of an ion
Peptides tend to fragment along the backbone.
Fragments can also loose neutral chemical groups like NH3 and H2O.
H...-HN-CH-CO . . . NH-CH-CO-NH-CH-CO-…OH
Ri-1 Ri Ri+1
H+
Prefix Fragment Suffix Fragment
Collision Induced Dissociation
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Ionisation
Proteins Polar Nonvolatile Thermally unstable
Ionisation transfers analyte into gas phase No degradation
Matrix-assisted laser desorption ionization (MALDI) Laser nitrogen beam (soft) Matrix protection (Sinapinic acid)
Electrospray ionization (ESI) No fragmentation
MA
LDI
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Mass spectrometry
WE Stephens 1952 Patent
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Mass spectrometry
Analytical technique mass-charge ratio (m/z) charged particles
Ion source
Mass analyzer
Detector
”Mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.
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Mass analysers
Scanning and ion-beam mass spectrometers TOF and Q
Trapping mass spectrometers IT and Orbitrap
Whole protein mass analysis time-of-flight (TOF) MS, or Fourier transform ion cyclotron resonance (FT-ICR).
Mass analysis of proteolytic peptides more popular Low costs Sample preparation is MALDI time-of-flight instruments Multiple stage quadrupole-time-of-flight and quadrupole ion trap also find use in this application.
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Mass spectrometers
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Base peak chromatogram
Yates JR, et al. Annu Rev Biomed Eng. 2009
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Mass chromatogram
Yates JR, et al. Annu Rev Biomed Eng. 2009
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Quantitative analysis
Yates JR, et al. Annu Rev Biomed Eng. 2009
Isot
opic
labe
ling
Labe
l-fre
e an
alys
is
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Quantitative proteomics
Yates JR, et al. Annu Rev Biomed Eng. 2009
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Protein identification
Single-step?
Components for separating identifying and quantifying the polypeptides tools for integrating and analysing all the data
Two main tracks. 2DE & MS Limited protein purification & peptide MS/MS
+/- isotope tagging
Efficient MS identification of gel-separated proteins Peptide-mass mapping by MALDI-TOF Peptide sequencing by ESI-MS/MS
Yates JR, et al. Annu Rev Biomed Eng. 2009
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Thank youDimitri Raptis & Alexander Koegel
www.draptis.eu/proteomics.ppt
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ProteomicsPost-translational modifications using mass spectrometry
www.draptis.eu/proteomics.ppt
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Interactions & modifications
Patterson & Aebersold, Nature Genetics, 2003
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Post-translational modifications
Any modification to the translated form of a protein Covalent additions that regulate protein function determining their activity state, cellular location dynamic interactions with other proteins
Analysis is challenging Determination -> insight into biological function
The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins mass spectrometry
Mann & Jensen. Nature Biotechnology 2003
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Common PTMs
Mann & Jensen, Nature Biotech, 2003
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Protein phosphorylation
Post-translational modification of proteins serine, threonine tyrosine residue is phosphorylated by a protein
kinase by the addition of a covalently bound phosphate group.
Phosphoregulation Phosphorylation -> most common
regulation of protein function Switches between
phosphorylated -> unphosphorylated
active -> inactive
Mann & Jensen, Nature Biotech, 2003
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Functions of phosphorylation
"activate" or "energize" a molecule to participate in a reaction with a negative free-
energy change.
Inhibit its activity Tyrosine kinase called "src” it folds on itself Masks its own kinase domain, and is thus shut
"off".
Mann & Jensen, Nature Biotech, 2003
Bound to other proteins which have "recognition domains” Distinct signaling system may be activated or inhibited
Degraded by the ATP-dependent ubiquitin/proteasome pathway substrates for E3 ubiquitin ligases only phosphorylated
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Glycosylation
Glycosylation is the addition of saccharide to a protein or a lipid molecule
N-Linked Glycosylation
Amide nitrogen of Asparagine
O-Linked Glycosylation
Hydroxy oxygen of Serine and Threonine
Mann & Jensen, Nature Biotech, 2003
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Additional modifications
Proteins subjected to: methylation acetylation glycosylation oxidation nitrosylation or ALL of these modifications,
time-dependent combinations potential complexity one has to deal with when studying protein
structure and function
Jensen, Nature Reviews, 2006
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Challenging analysis
“Global proteomics” methods do not work to find PTMs A few modifications can be programmed into search programs –
destroy MS speed and accuracy PTMs are usually present at too low a concentration. Only the
unmodified version will be seen
Affinity enrichment techniques are required Affinity columns (eg., lectin cloumns, IMAC) Antibodies Tags (e.g., biotin)
Jensen, Nature Reviews, 2006
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Mapping strategy - MS
Use MS to determine PTM of isolated protein
Enzymatic or chemical degradation of modified protein
HPLC separation of peptides
MALDI and/or ESI used to identify PTM
MS/MS used to determine location of PTM(s)
Mann & Jensen, Nature Biotech, 2003
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Assigning PTMs - MS
Jensen, Nature Reviews, 2006
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Thank youAlexander Koegel & Dimitri Raptis
www.draptis.eu/proteomics.ppt