Proteomics

Dimitri Raptis & Alexander Kögel Protein identification using mass spectrometry www.draptis.eu/proteomics.ppt

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

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.

Amino acid sequence

” Proteins." Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.

Protein structure

Weaver, Molecular Biology, McGraw Hill: Boston, 2002

Proteome

 Entire set of proteins expressed by     Genome Cell Tissue Organism > 400.000 proteins, dynamic

Human proteome

Aebersold et al, Systems biology - ETH Zurich, https://www.e-pics.ethz.ch/

Why proteomics?

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

Multidisciplinary

Cells, tissue HPLC MALDI, ESI TOF, Q, IT Algorithms

Typical stages

Aebersold R, Mann M, Nature. 2003

Quantification strategies

Domon B, Aebersold R. Science. 2006

Top down or bottom up?

 Bottom-up  Most common   Starting with proteolytic fragments Piecing the protein back together  de novo repeat detection Fragment ions of peptides MS/MS Proteolytic digest e.g. Trypsin  Protein Top down    Tandem MS of whole protein ions  Pulling them apart Electron capture dissociation Extensive sequence information MS/MS Fragment ions of protein ” Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.

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

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

Robotic isolation

” Protein mass spectrometry" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.

Shotgun proteomics

HPLC

” HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.

HPLC

 HPLC/MS  Peptides from protein mixture fractionated in steps  Eluent   ESI-MS MALDI: series ” HPLC" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc.

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.

Proteins

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Peptides

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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

Collision Induced Dissociation H + H...-HN-CH-CO . . . NH-CH-CO-NH-CH-CO-…OH R i-1 R i R i+1

Prefix Fragment Suffix Fragment Peptides tend to fragment along the backbone.

Fragments can also loose neutral chemical groups like NH 3 and H 2 O.

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

Mass spectrometry

WE Stephens 1952 Patent

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.

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.

Mass spectrometers

Base peak chromatogram

Yates JR, et al. Annu Rev Biomed Eng. 2009

Mass chromatogram

Yates JR, et al. Annu Rev Biomed Eng. 2009

Quantitative analysis

Yates JR, et al. Annu Rev Biomed Eng. 2009

Quantitative proteomics

Yates JR, et al. Annu Rev Biomed Eng. 2009

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

Thank you

Dimitri Raptis & Alexander Kögel

www.draptis.eu/proteomics.ppt

Proteomics

Alexander Kögel & Dimitri Raptis Post-translational modifications using mass spectrometry www.draptis.eu/proteomics.ppt

The Mass Spectrometer (MS)

 Moving ions are separated in a magnetic field according to their mass-charge-ratio

Importance

for modern Proteomics

 Molecular weight determination of peptides and proteins  Amino acid sequencing of peptides  Detection of post-translational modifications (ptm)

Protein phosphorylation

 Phosphoregulation  Common regulation of protein function  Phosphorylaion -> Activation  Mostly Ser, Thr, Tyr are phosphorylated by protein kinases Mann & Jensen, Nature Biotech, 2003

Identification of a Phosphorylation-site 1.

Isolation of known protein 2.

Enrichment with Metal Affinity Complexation 3.

Digestion with Trypsin

4.

Mass Spectrometry with peptides

Identification of a Phosphorylation-site  Modification is proven to be in a peptide of certain mass

Identification of a Phosphorylation-site  Further fragmentation of peptide in collision chamber  Second analysis with MS and comparison to databank will lead to a shifted aa in the MS screen

Most studied PTMs

Thank you

Alexander Kögel & Dimitri Raptis

www.draptis.eu/proteomics.ppt