Protein Seperation Methods

Protein techniques Protein Identification Protein Expression Protein Purification Protein-Protein interactions Application in literature

Protein Identification Sequencing (Edman degradation) Determine approx the first 20 AA Centrifugation (cellular location) 1D/2D Gel Electrophoresis Mass spectrometry Break sample into peptides Molecular mass is determined using mass-to-charge ratios of ions AA sequence can be determined

Centrifugal separation Differential centrifugation Sediment coefficient (mass, density and shape) Crude separation of cell fractions Rate Zonal (mass) Density of particles > density of solution Separation based on rate of sedimentation Time sensitive Isopycnic (density) Density of particles < highest density of solution Separation based on reaching equilibrium position in density gradient Time in-sensitive

Differential centrifugation

Gradient centrifugation

Gel electrophoresis Denaturing – SDS-PAGE Non-denaturing SDS gives uniform neg. charge Separates proteins by size/mass Non-denaturing Separates based on charge and size/conformation Often combined with Western blotting (using antibodies specific for proteins of interest)

SDS-PAGE

2D gel electrophoresis 1st dimension 2nd dimension Separation based on pI isoelectric focusing of zwitterions 2nd dimension Normal SDS-PAGE

2D-GE

Difference Gel Electrophoresis (DiGE)- quantitative comparisons

Mass spectrometry IDs based on mass-to-charge ratio Samples are broken down and analyzed Proteins -> peptides Able to determine seq of peptides Database search to ID protein

Mass spectrometry

Mass spec combos LC/MS MALDI-TOF MS Tandem MS Liquid chromotography to separate peptides MALDI-TOF MS Matrix-assisted laser desorption-time of flight Samples are ionized and “flight time” through an electrified tube is measured Tandem MS Multiple MS measurements on a single sample Identifies peptide sequence

Protein Expression and Purification Why? Obtain pure (clean) protein "Don't waste clean thinking on dirty enzymes“ - Arthur Kornberg Powerful experimental tool Simplifies the system in which you are asking a question Confirmation of a hypothesis that is developed in a more complex system Arthur Kornberg "Don't waste clean thinking on dirty enzymes"

Protein Expression Why over-express the protein? Make large quantities to facilitate purification/study Analyze biochemical properties Perform structural analyses Crystallization NMR Identify protein interactions Make Antibodies

Expression systems E. coli Yeast Insect cells In-vitro systems Prokaryotic expression workhorse Yeast For bacterial or eukaryotic proteins Large amounts of protein Insect cells Post-translational modifications In-vitro systems wheat germ, rabbit reticulocyte

Purification strategies Exploiting protein chemistry Size/Mass Charge Hydrophobicity Antibody affinity Protein Tags Often used in combination

Size Exclusion Chromatography Separation based on size of protein

Ion exchange & hydrophobicity Non-tagged proteins Separation based on charge or degree of hydrophobicity Bound proteins are eluted with salt containing buffers

Ion exchange

HPLC High performance (pressure) liquid chromatography Sample is passed over column of varying hydrophobic nature- more hydrophobic particles bind tighter and elutes later. Eluate is analyzed by a detector UV, refractive index, fluorescence Can be combined with mass spec (LC/MS)

HPLC

Affinity/Ab columns Purify tagged proteins Interaction between two molecules Solid phase- immobilized on column Mobile phase- binds while passing over column Buffer conditions regulate binding & dissociation pH, ionic strength, competing

Ab affinity column Include Protein A-agarose/sepharose

Tagging the protein Clone gene in frame with a unique protein sequence or “tag” Advantages Purification Use tag to selectively remove protein from a complex sample Protein visualization/tracking Fluorescent protein tags, labeled antibodies

Protein Tags His GST S-Tag, C-myc, HA, flag small 6 HIS residues bind to nickel columns GST Binds to glutathione resin/beads S-Tag, C-myc, HA, flag Antibody affinity columns

Protein Tags 6xHistidine binds metal chelating resin- Cu2+, Ni2+, Co 2+

What can you do with purified proteins? Biochemical and functional characterization DNA binding, enzyme activity, stability, etc., Structural analyses informs function NMR, crystallography, circular dichroism Study protein-protein or protein-DNA interactions Develop antibodies

Structural Analyses Circular dichroism (basic 2o structure) Nuclear Magnetic Resonance Protein Data Bank- pdb.org X-ray Crystallography Electron Microscopy Using structure to inform drug design/mutagenesis

Using multiple fragment binding in an enzyme active site to determine possible directions of “growth chemistry” within the active site.

Structure of the final inhibitor (IC50 = 3.7 nM) Fragment linking: X-ray crystal structure of fragments binding at different sites of thrombin S2–S4 sites (IC50 = 12 μM) S1 site (IC50 = 330 μM) Structure of the final inhibitor (IC50 = 3.7 nM)

Protein-protein interactions Two hybrid system Co-immunoprecipitation Surface plasmon resonance Protein arrays Protein crosslinking FRET- fluorescence Resonance Energy Transfer

Immunoprecipitation Specific Ab binds protein in solution Solution is eluted over Protein A column Protein is eluted from Ab Co-IP Also allows for study of proteins bound to IP’d protein ID protein complexes

Isolation and identification of protein binding partners

SPR Surface plasmon resonance Biomolecular interaction analysis BIACORE Protein is immobilized onto surface Light is refracted onto thin metal layers Immobile protein refractive index changes when ligand is bound

Protein arrays ELISA based format: Ab, proteins, peptides immobilized Solution to be searched is layered on top Binding of partner proteins is detected by SPR or fluorescence

Protein crosslinking X-linking agent “locks” interacting proteins Formaldehyde Linking is highly specific Can be performed in vivo Cell extract can be subjected to IP assays Identify x-linked proteins via co-IP Chromatin IP – ID bound DNA sequences

Fluorescent protein tags Protein-protein interactions with fluorescence energy transfer (FRET) Visualizing protein localization Green fluorescent protein (GFP)