methods in protein analysis biochemistry

8/11/2019 Methods in Protein Analysis Biochemistry

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Methods in Protein Biochemistry


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Gel Electrophoresis


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Polyampholyte Character of aTetrapeptide and Isoelectric Points

Isoelectric Point (pI) , pH at

which molecule has net zerocharge, determined usingcomputer program for knownsequence or empirically (byisoelectric focusing).

Group pKaa -NH 3+ 9.7 Glu g-COOH 4.2Lys e -NH 3+ 10.0a -COOH 2.2


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Salting Out: Ammonium SulfatePrecipitation in Protein Fractionation


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Centrifugation

Centrifugation Methods

Differential (Pelletting) simple methodfor pelleting large particles using fixed-angle rotor (pellet at bottom of tube vs.supernatant solution above)

Zonal ultracentrifugation ( e.g. , sucrose-gradient) swinging-bucket rotor

Equilibrium-density gradientultracentrifugation ( e.g. , CsCl) swinging-bucket or fixed-angle rotor

Low-speed, high-speed, orultracentrifugation: differentspin speeds and g forces


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Zonal Centrifugation: Sucrose-GradientPreparative Ultracentrifugation

Separates by sedimentation coefficient (determined

by size and shape of solutes)


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Sucrose-Gradient PreparativeUltracentrifugation


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Equilibrium Density GradientUltracentrifugation

Used in Meselsen-Stahl experiment. Separates based on densities of solutes. Does not require premade gradient. Pour dense solution of rapidly diffusing substance in

tube (usually CsCl). Density gradient forms during centrifugation (self -

generating gradient).

Solutes migrate according to their buoyant density(where density of solute = density of CsCl solution).


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Column Chromatography

Flow-through Eluate


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Different Types of Chromatography

Gel filtration/size exclusion/molecular sieve - separates by size(molecular weight) of proteins

Ion exchange (cation exchange and anion exchange) -

separates by surface charge on proteins Cation exchange: separates based on positive charges of

solutes/proteins, matrix is negatively charged Anion exchange: separates based on negative charges of

solutes/proteins, matrix is positively charged

Hydrophobic interaction - separates by hydrophobicity ofproteins Affinity - separates by some unique binding characteristic of

protein of interest for affinity matrix in column


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Ion-Exchange Chromatography


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Gel Filtration Chromatography


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Affinity Chromatography


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Cleavage of Polypeptides for Analysis

Strong acid ( e.g. , 6 M HCl) - not sequence specific

Sequence-specific proteolytic enzymes (proteases) Sequence-specific chemical cleavage ( e.g. ,

cyanogen bromide cleavage at methionine residues)


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Protease Specificities


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Cyanogen Bromide Cleavage atMethionine Residues


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Protein Sequencing: Edman Degradation

PTH = phenylthiohydantion

F3CCOOH = trifluoroacetic acid

PTC = phenylthiocarbamyl


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Identification of N-Terminal Residue

Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildlyacidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase).

NO 2


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Separation of Amino Acids by HPLC


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Protein Identification by MassSpectrometry


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Protein Identification by MassSpectrometry

Two main approaches:

1. Peptide mass fingerprinting: Proteolytic digestion of protein,then determination m /z of peptides by MS ( e.g. , MALDI-TOFor ESI- TOF), search fingerprint against database. Successof ID depends on quality/ completeness of database forspecific proteome.

2. Tandem MS (MS/MS e.g. , nanoLC-ESI-MS/MS):Proteolytic digestion of protein, separation and determinationof m /z of each (MS-1), then determination of collision-induceddissociation fragment spectrum for each peptide (MS-2).Gives context/sequence-dependent information, so more of ado novo sequencing method.


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Locating Disulfide Bonds

O

O -I

iodoacetate


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Determing Primary Structure of an EntireProtein


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Reactions in Solid-Phase PeptideSynthesis


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methods in protein analysis biochemistry

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