Proteomics

  • Proteomics

    • It is the large-scale study of proteins, particularly their structures, functions and interactions

    • Proteins are vital parts of living organism as they are the main components of the physiological metabolic pathways of cells

    • The proteome encompasses the proteins of an entire system

      • Cell, Tissue or Organism

Including modification

  • The proteome is dynamic

    • It varies with

      • Time

      • Responses to the environment

      • During development and differentiation

 

 

 

  • Complexity of the proteome

    • Genome : 20 - 25000 genes

      • Population variations in alleles

    • Mrna

      • Estimated that 40-60% can be alternatively spliced

    • RNA editing

      • Mainly Cytidine> Uracil

      • Adenine>Inosine

    • RNA splicing estimates vary

RNA editing: C>U - prime example is Apolipoprotein B: intestine produces a transcript with a CAA edited to UAA (stop codon), yielding a shorter form than the liver produces.

  • PTMs

    • Cleavage

    • Phosphorylation

    • Acetylation

    • Glycosylation

    • Lipidation

    • (30+ PTMS reported)

  • Protein-protein/protein-nucleic acid interactions

    • Receptors

    • Metabolic pathways

    • Signalling complexes

    • Transcription activation

 

  • Comparing proteomes

    • Proteome size in that

      • Cell

      • Tissue

      • Organism

    • Comparison with proteome

      • Under different conditions

      • After differentiation

      • Tumour stage

      • Drug treatment

    • Changes in protein

      • Abundance

      • Activity

      • Modification

      • Interactions

      • Locations

 

 

  • Comparing proteomes

    • Data mining to find relevant links (guilt by association)

      • All proteins which show alteration - statistical rigour

      • Proteins associated with a relevant

        • Protein complex

        • Signal transduction pathway

        • Product of shared transcriptional regulation

 

 

 

  • Methods for exploring the proteome

    • Classical Protein Biochemistry

      • Purification

      • Activity

      • Structure determination

    • SDS-Polyacrylamide gel electrophoresis

    • Two dimensional gel electrophoresis(size and charge)

      • Differential 2D gel electrophoresis

 

 

 

 

  • Immunoaffinity techniques

    • Localisation/Co-localisation

    • Purification

      • Single proteins

      • Functional groups

      • Complexes

 

 

 

 

  • Protein-protein interactions

    • Tandem-affinity tags for protein complex analysis

      • Tag the DNA sequence of a protein of interest with a TAP-tag

      • Express fusion protein in cells of interest

      • Purify

      • Identify associated proteins

      • Repeat using DNA sequences of purified proteins

      • Build associated network

      • Tap-Tags are one of many ways proteins can be tagged with other molecules to help isolate or report on interaction partners. 

      • Other options include yeast two-hybrid and associated techniques

Tap tagging can be done on a large scale

 

 

 

 

 

 

  • How can we identify proteins

    • Historically by extensive purification and characterisation

      • Amino acid sequencing

    • Now - Mass spectrometry methods

      • Direct identification/sequencing of purified protein fragments

    • From mixtures of proteins

      • Require existing knowledge of protein sequences

      • Has benefitted from

        • Genome initiatives

        • Bioinformatics of protein coding regions

        • Sequence databases

 

 

 

  • Protein sequence databases

    • Sites

      • Expasy - Geneva

      • European Bioinformatics Institute - EBI; Cambridge

      • NCBI - Bethesda, USA

    • Content

      • Directed sequenced protein data

      • Protein sequence derived from Genomic/mRNA sequences

      • Variants

      • Modifications

    • Key points

      • Comprehensive and curated

      • Non-redundant

      • Taxonomic

      • Internet accessible/searchable

 

 

  • Protein  Mass Spectrometry - has two key methods

 

Electrospray Ionisation (ESI)

Matrix-assisted, laser desorption/Ionisation(MALDI)

John B. Fenn

Kochi Tanaka

2002 Nobel prize in Chemistry

2002 Nobel prize in Chemistry

Analysis of ionised peptide/proteins

Analysis of ionised peptide/proteins by mass spectrometry

Applying a high voltage to an aerosol nozzle through which protein solution is passed

Protein/Peptides dried with an acid matrix compound

Solvent evaporates in vacuum of mass spectrometer, leaving charge on the biomolecules

UV laser light causes ablation(desorption) of matrix and peptides

Very good for large molecules

Matrix helps transfer protons(+-H) to the peptides

Gives high quality information when combined with Tandem mass spectrometry(ESI-MS-MS)

Ionised peptides can be examined for mass to charge ratio(m/z) in a time of flight spectrometer(TOF)

Can de novo sequence peptides

 

Predominantly multiple ionisations

Predominantly single ionisation

 

 

 

 

  • Mass Spectrometry Terms

    • Mass analysers

      • Time of flight(TOF) mass analyser

        • Accelerates charged ions in a vacuum tube, measuring the flight time

      • Quadrupole mass filters

        • Uses radio frequencies and DC voltages to filter ions  based on their mass to charge(m/z) ratio

    • Collision cell

      • A device which enables collision activated dissociation of peptides into smaller fragments

      • Usually by collision with an inert gas

    • Ion optics

      • Ion lenses

        • Focus ions into an appropriate beam

      • Reflection

        • Mirror for ions, used to extend flight time

      • Ion detector

        • Detects ions striking it, amplifies signal

      • Ion Traps

        • Similar to quadrupole filters, but capable of trapping and accumulating a chosen ion

 

 

 

 

 

 

  • Automation : LC-MS/MS

    • Whole proteomes are complex mixtures

      • Increasingly so when Trypsin is fragmented

      • Too many ions

      • Pre-sort ions using Liquid chromatography(LC)

        • Ion exchange column

          • Charge

        • Reverse-phase column

          • Hydrophobicity

        • Can be automated

      • Double sort

        • Allowing direct elution from the second column into the MS

      • Extracted ion chromatograms(XICs)

 

 

 

 

  • How can we use MS to identify proteins

    • Many proteins are too big to identify by MS

    • Enzymatic proteolysis gives peptide fragments

    • Trypsin is cheap and reliable

    • Cleaves c-terminal to Arginine(R ) and Lysine (K)

    • Except when Arg (R ) is N terminal to Proline (P)

    • Exact peptide size includes abundance of natural isotopes

    • 3-4 decimal places is needed in m/z

 

 

 

 

  • Identification of proteins from peptide m/z ratios

    • Databases exist containing the theoretical cleavage fragments of all known proteins with trypsin and other enzymes

    • Accurate peptide m/z ratio is present for each peptide in DB

    • Comparison of your peptide(s) gives a list of possible matches per peptide

      • Less hits with more accurate m/z ratios

    • Multiple peptides giving identity with the same protein support good quality identification

    • Problems were few peptide m/z's

    • Problems with common motifs

      • Found in multiple proteins

 

 

  • Sequencing peptides by MS-MS

    • Pick one peptide seen by MS at a time

      • Select for that peptide using quadrupole filter/ion trap

      • Collide it with inert gas

      • Peptide bond breaks

      • Fragments are varies proportions of the whole peptide

      • Analyse the fragments by TOF mass analysis

      • Use amino acid masses to calculate sequence

      • 2 ion series

        • Y series (charge on C terminus) and

        • B series(charge on N terminus)

 

 

Y series ions shown

 

  • Identification of proteins from peptide sequence

    • Databases exist containing the 'theoretical' cleavage fragments of all known proteins with trypsin(and other enzymes)

    • Can examine all databases for possible coding sequence matches

    • Number of hits decreases and confidence increases with added peptide sequences of same protein

    • Problems where only one peptide sequenced

    • Problems with PTMs

 

 

 

  • Quantitative proteomics

    • MS is inherently non-quantitative

    • Quantitative analysis by multiple methods

    • Stable isotope labelling by amino acids in cell culture(SILAC)

    • Isobaric tags for relative and absolute quantitation(iTRAQ)

      • Isobaric(same weight) - nominally same mass

    • iTRAQ as an example

 

 

 

 

 

 

 

 

 

 

  • Applications and the future

    • Protein interactions

    • Mapping of entire cellular systems

    • Drug effects

    • Disease biomarkers

    • Rapid diagnostics

      • From breath or other sample

    • Modelling biological responses

    • Improved specificity drugs

    • Improved understanding off complex diseases