Purification and Primary Sequence Determination

Formation of Peptides

peptides are small condensation products of amino acids

Peptides

linear sequence of amino acids with one free N terminus and one free C terminus held together by peptide bonds

peptide bonds have partial double bond characteristic due to resonance

sequences are written from the N to C terminus

Covalent Structures of Proteins

1. gene sequence - each amino acid is coded for by 3 nucleotides; DNA sequence can be translated into protein sequence

2. chemical and enzymatic sequencing of purified protein, mass spectrometry sequencing

Protein Purification

two options:

1. know gene sequence, protein sequence, size + charge + maybe something about the structure and function, design a purification scheme based on those properties

2. know protein function, don’t know sequence, don’t know size + charge + something about the structure, try different techniques and monitor protein concentration and function

separation relies on differences in physio-chemical properties

• size

• charge

• affinity for a ligand

• solubility

• hydrophobicity

• thermal stability

chromatography is commonly used for preparative separation

Protein purification needs…

an assay for function and an assay for protein

UV absorbance by the Beer-Lambert Law for the presence of protein

Separation Based on Solubility

ammonium sulfate precipitation: as the ionic strength of the solution increases, water molecules get “tied up” with hydrating the ions and no longer hydrate the dissolved protein → precipitation

larger protein surface = lower ammonium sulfate concentration at which it will precipitate

small polar molecules will have stronger ionic strength

• frequently the first step in purification because it removes large fractions of contaminating proteins

• not very specific (only solubility matters), proteins require further purification

at pH = pI proteins will usually precipitate because loss of charge-charge repulsion (less solubility)

Separation Based on Size

gel filtration chromatography

size exclusion chromatography

each size exclusion resin has a specific range of sizes (300-15000, 50000-500000, 10000-100000)

• protein below the limit will all elute together at the bed volume and proteins greater than the limit will elute together at the void volume

Charge

pI of a protein is the pH at which the protein had no net charge → can be determined experimentally

if pI is less than pH the protein is NEGATIVELY charged

if pI is more than pH the protein is POSITIVELY charged

charge on protein depends on pI and pH of the buffer in ion exchange resins

anion exchange resins: DEAE sepharose, Q sepharose, bead-linker quaternary amine

cation exchange resin: CM CH2-COOH, SP CH2SO3H

Affinity

ATP columns are used to purify any protein that binds to ATP or any of the nucleotide cofactors

metal binding proteins or His rich proteins bind to Ni columns

• ATP is covalently linked to the column - any protein that binds to ATP will bind to the column and will constantly come off + rebind

• add ATP to the column buffer to get the protein off the column

• the tighter the protein binds to the column the more free ATP in the buffer it takes to get protein off

Polyacrylamide Gel Electrophoresis

separates proteins based on size

samples are boiler in detergent SDS containing DTT to denature protein and break disulfide bonds → multi-subunit proteins are separated into individual chains

all proteins are coated in SDS so they are negatively charged regardless of amino acid composition

SDS PAGE: Molecular Weight

SDS = sodium dodecyl sulfate - a detergent

SDS micelles bind to and unfold all the proteins, gives all proteins a uniformly negative charge (native shape doesn’t matter)

rate of movement will only depend on size

SMALL PROTEINS MOVE FASTER - too thick = nothing moves, too thin = everything moves too much

Quantitative Amino Acid Analysis

gives basically all the amino acids in the protein but no sequence information

1. hydrolyze all amide bonds for 12-36 hours in sealed tube

2. run the mixture on an ion exchange column to isolate

3. amino acids are derivatized with a chromophore (DABS) before or after the column is run

asn and gln will not show up because they have amide bonds that get hydrolyzed to glu and asp

End-Group Analysis

to determine the identity of the amino acid at the N or C-terminal of a peptide/protein and the # of polypeptide chains in the protein

dansyl chloride and FDNB react with free amino groups, also lys side chain

standards can be used to identify dansyl or DNP labelled amino acids

2+ amino acids indicate heterogenieity (one peak) or more than one polypeptide chain in the protein (multiple peaks)S

Disulfide Bond Cleavage

cysteine residues can interfere with sequence determination due to disulfide bonds

beta-mercaptoethanol or DTT (makes cyclic disulfide) break disulfide bonds by disulfide exchange - making intermolecular bonds within molecule to break the ones from protein

carboxymethylation with iodoacetate prevents disulfide bonds from reforming

Protein and Peptide Sequencing

only reliable for 30-50 amino acids in a sequence; method has to break big proteins into smaller, specific peptides for sequencing

chymotrypsin and trypsin are specific proteases to make smaller pieces

1. reduce and alkylate disulfide bonds (hydrolyze + reduce)

2. cleave polypeptide to smaller fragments by 2 or more methods e.g. trypsin and cyanogen bromide (cleaves at MET)

3. sequence smaller fragments e.g. Edman degradation

4. overlap sequences to determine overall sequence

5. repeat without reduction of disulfides to locate position of bridges

Edman Degradation Method

makes amino acid sequence from the N-terminal end but each step is only 99% efficient

after 20-50 you get too much noise and can’t differentiate between what you just cut

cannot sequence through disulfide bonds → need to reduce disulfide bonds to Cys first

Putting Everything Together

1. purify protein

2. treat with beta-mercaptoethanol or DTT and iodoacetate if needed

3. treat separately with trypsin, chymotrypsin and/or CNBr

4. separate the peptides by ion exchange or reverse phase HPLC

5. sequence each peptide by the Edman degradation method

6. analyze data to generate intact peptide

Protein Sequencing by Mass Spectrometry

• protein is cleaved into peptides, usually with trypsin since it generates and fragments

• fragments are subjected to tandem MS-MS by electrospray ionization

• patterns of fragments are analyzed and compared to known sequences

• sequence is generated

What can sequences do for you?

• identify the target protein by comparison to a similar, known protein to predict new protein’s function

• allows to get a crystal structure to get a hypothetical structure

• can show how related a particular protein is to the same protein from other organisms by looking at primary sequences