ProteomicsProtein 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

TOF, Q, ITMALDI, ESIHPLCCells, tissue Algorithms

Typical stages

Aebersold R, Mann M, Nature. 2003

Quantification strategies

Domon B, Aebersold R. Science. 2006

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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

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”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 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

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

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

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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 youDimitri Raptis & Alexander Koegel

www.draptis.eu/proteomics.ppt

ProteomicsPost-translational modifications using mass spectrometry

www.draptis.eu/proteomics.ppt

Interactions & modifications

Patterson & Aebersold, Nature Genetics, 2003

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

Common PTMs

Mann & Jensen, Nature Biotech, 2003

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

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

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

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

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

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

Assigning PTMs - MS

Jensen, Nature Reviews, 2006

Thank youAlexander Koegel & Dimitri Raptis

www.draptis.eu/proteomics.ppt