How to study a protein

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How to study a protein ?
Expressed
Tissue

• Old way
• from tissues
• New way
• over-expression in micro organisms
• plasmids in bacteria
• baculovirus vector in insect cells

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Expression of Recombinant Proteins

• Expression Vector Features
• gene dosage
• strong promoter
• inducible promoter
• translation signals, etc
• fusion proteins
• protease defective hosts

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Expression vector
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Preparation of Expression Vector

• subclone insert from current vector to expression
  vector
• design PCR primers to amplify region of interest
• expressed protein must be
• correct orientation
• in-frame

BamH1 EcoR1 SmaI
SalI XhoI NotI ... ATC GAA GGT CGT GGG
ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC
... ... TAG CTT CCA GCA CCC TAG GGG TCC TTA AGG
GCC CAG CTG AGC TCG CCG GCG ... ... Ile Glu Gly
Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser
Gly Arg ... Factor Xa
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• increase stability
• affinity purification
• detection/assay
• spectrophotometric
• binding assays
• antibodies
• export signals

Fusion Proteins
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Transfection Methods

• calcium phosphate
• DEAE-dextran
• electroporation
• liposomes
• protoplast fusion
• ballistics (gene gun)
• microinjection

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• Possible IB Cure
• isolate by differential centrifugation
• solubilize in urea
• re-nature protein (?)

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Problems with Expression of Eukaryotic Proteins
in Prokaryotes

• stability (protein and gene)
• proper folding and disulfide formation
• post-translational modifications
• asking species specific questions

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• Post-translational modifications
• Phosphorylation
• Ubiquitination (proteasomal degradation)
• Methylation
• Acetylation
• Glycosylation (N-linked, O-linked)
• Oxidation
• Nitrosylation, etc.

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Eukaryotic Expression Systems

• in theory, plasmids can be introduced
• into any host
• yeast are easy to maintain in lab
• Saccharomyces cerevisiae
• Pichia pastoris
• viruses
• several mammalian
• baculovirus (insect)

vaccinia (lytic) adenovirus (lytic) papilloma
(episomal) retrovirus (integrated)
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Protein Purification Techniques Large Scale

• Salting out
• Dialysis
• Gel-Filtration Chromatography
• Ion-Exchange Chromatography
• Affinity Chromatography
• High-Pressure Liquid Chromatography

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Differential centrifugation as a first step in
protein purification
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Gel-Filtration Chromatography Separation based
on size
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High Pressure Liquid Chromatography
High pressure limits diffusion and increases
interactions with chromatography media HPLC gives
very high resolution of protein components
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Ion-Exchange Chromatography Separation based on
charge
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Types of ion-exchange chromatography media
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Affinity Chromatography
Small molecules are attached to beads and complex
protein mixtures are applied. Bound proteins
can be eluted with the small molecule or with
denaturing reagents (urea, guanidine, etc.)
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Elution fractions from affinity chromatography
Fraction number
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pure protein
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1. Engineered protease site allows removal of
fusion partner
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2. Addition of a few residues should have minimal
effect on recombinant protein

• His6 Tag
• add 6 consecutive His to either end
• binds metals

• Epitope Tag
• 6-12 amino acids
• mAb for detection or purification

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Mass spectrometry of Proteins
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What is a mass spectrum?
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MS of proteins is used for

• Identification of proteins
• Exact mass of a protein
• Peptide mass fingerprint
• Aminoacid sequence
• Protein characterization
• Posttranslational modifications
• De Novo sequencing
• Analysis of complex protein mixtures
• Quantification of compounds

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How does it work?

• Sample preparation
• Isolation, purification, de-salting
• Digestion with selective proteolytic enzyme
• Ionisation
• MALDI (Matrix Assisted Laser Desorption
  Ionisation)
• Electrospray
• Ion detection and mass measurment

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Samples

• Protein solutions, protein mixtures,
• bands from SDS PAGE, 2D Gel spots
• Whole protein
• Protein digest

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Trypsin digestion

• Trypsin is a selective proteolytic enzyme
• Cuts after a Lys (K) and after Arg (R) aminoacid
  residue

Denaturation (Heat, SDS)
Trypsin digestion 12h _at_ 37C
Protein
Peptides
MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKE
KVNELSK DIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKE
DVPSERYLGYLEQLL RLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKE
PMIGVNQELAYFYPELFRQFYQL DAYPSGAWYYVPLGTQYTDAPSFSDI
PNPIGSENSEKTTMPLW
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Trypsin digestion

• Trypsin is a selective proteolytic enzyme
• Cuts after a Lys (K) and after Arg (R) aminoacid
  residue

Denaturation (Heat, SDS)
Trypsin digestion 12h _at_ 37C
Protein
Peptides
MK LLILTCLVAVALAR PKHPIKHQGLPQEVLNENLLR
FFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIK
QMEAESISSSEEIVPNSVEQK HIQK EDVPSER
YLGYLEQLLR LK K YK VPQLEIVPNSAEER LHSMK
EGIHAQQK EPMIGVNQELAYFYPELFR
QFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK
TTMPLW
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Ionization

• 2 methods used in protein MS
• MALDI Electrospray

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MALDI
Matrix Assisted Laser Desorption Ionization
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MALDI
Matrix Molecules

• Acidic- act as a proton source to encourage
  ionization of the analyte
• Have a strong optical absorption in the UV, so
  that they rapidly and efficiently absorb the
  laser irradiation
• Give the analyte molecule a single, sometimes
  double charge

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Electrospray

• No matrix necessary
• Analyte in acidic solution
• gains a positive charge (H)
• Positive ions form
• spray in electric field
• Produces multiple
• charged ions

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Electrospray
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How a mass spectrum is created

• ToF Time of Flight
• Measures time from ion entry to the flight
  chamber to its detection on the detector
• Light ions are fast, more massive ions are slower
• A double charged ion is 2x faster

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Time of Flight
Start Detector
1x 1x 2x
The mixture of ions is separated by their
velocity in electric field
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Time of Flight
Start Detector
1x 1x 2x
The measured mass spectrum is a ratio of mass to
charge, m/z
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Mass spectrum
Ion intensity
Mass/charge
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Protein Identification
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Peptide Mass Fingerprint

• Trypsin cleaves the protein in defined positions
  selective digestion
• The resulting peptides
• can be predicted (software)
• are characteristic for a protein
• their masses (m/z) can be found in protein
  databases
• PMF can be used to identify a known protein

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MS peptide sequencing

• Fragmentation of a peptide precursor by
  collision with atoms of inert gas Argon
• Fragmenting occurs on defined sites of the chain
  the peptide bond
• The mass of the fragment is characteristic for a
  specific aminoacid
• Partial fragments can be used to identify a
  sequence

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WTF?
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Collision Induced Dissociation (CID)

• Peptide fragmentation
• precursor product ions

fragmentation
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Peptide sequencing

• Sequence AVDDFLISLDGTANK
• Database search
• BLASTp

Protein identification
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Protein identification

• Database search (Swissprot, NCBI, custom db)
• Known proteins, sequenced genomes, translated
  cDNA libraries, conserved sequences
• De Novo sequencing / BLASTp
• Unknown proteins, sequence homologies
• At least 3 peptides to identify a protein (PMF)
• At least 3 fragments to identify a peptide

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MS in combination with chromatographic separation
methods

• Gas chromatography (GC-MS)
• Not suitable for proteins
• Liquid chromatography (LC-MS)
• Wide selection of analytic columns
• C4 - C18 for proteins and peptides
• Hydrophilic Interaction for polar substances
• Allows pre-separation of analyzed mixture
• High sensitivity
• High resolution

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Appendix 1 1x charge vs 2x charge
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Appendix 2 b ions and y ions