PROTEIN SEQUENCING First Sequence The first protein sequencing was achieved by Frederic Sanger in 1953. He determined the amino acid sequence of bovine insulin Sanger was awarded the Nobel Prize in 1958 1
I. Strategy Determine number of polypeptide chains (subunits) Determine number of disulfide bonds (inter- and intrachain) Determine the amino acid composition of each polypeptide chain If subunits are too large, fragment them into shorter polypeptide chains Sequence each fragment using the Edman degradation method Complete the sequence by comparing overlaps of different sets of fragments II. End-group Analysis Number of chains can be determine by identifying the number of N- and C-terminal. N-terminal analysis Dansyl chloride Phenylisothiocynate (PITC)/ Edman reagent Aminopeptidase C-terminal analysis carboxypeptidase 2
N-terminal Analysis with Dansyl Chloride Main reagent: 1-dimethyl aminophthalene-5-sulfonyl chloride (dansyl chloride) Dansyl poplypeptide chain is prepared Acidic hydrolysis liberates all amino acid and the N- terminal dansyl amino acid Amino acids are separated Fluorescence of the dansyl amino acid is detected Type of aa is obtained from comparison with standard dansylated amino acids N-terminal Analysis Edman (Degradation) Nucleophilic attack on phenyl isothiocyanate (PITC), the Edman reagent, under mild alkaline conditions (Nmethylpiperidine/water/ methanol) Formation of a phenylthiocarbamyl derivative (PTC-peptide) 3
N-terminal Analysis Edman (Degradation) Anhydrous trifluoro acetic acid (TFA) is used to cleave the terminal amino acid in the form of a thiozolinone derivative leaving the other peptide bonds intact The thiozolinone (TZ) derivative is extracted in an organic solvent (e.g. N-butyl chloride) Peptide cleaved carries a free amino terminus N-terminal Analysis Edman (Degradation) The TZ is extracted into an organic solvent and treated with an acid (25 % TFA/water) to form phenylthiohydantoin (PTH) derivative PTH is detected from UV absorption at 296 nm 4
N-terminal Analysis-Edman Degradation PTH amino acid is separated from the other components by chromatography or electrophoresis The terminal amino is identified according to retention time or mass This sequence can be repeated to identify all amino acid in short peptide chains (40-60 amino acid long) Edman Degradation on Protein Sequencer Perkin Elmer Applied Biosystems Model 494 Procise protein/peptide sequencer http://www.biotech.iastate.edu/facilities/protein/nsequence494.html 5
Edman Degradation on Protein Sequencer By-products of Edman Degradation 6
N- and C-terminal Analysis-Exopeptidase Method Exopeptidases cleave the terminal residue of a polypeptide chain Aminopeptidases cleave the N-terminal residues Carboxypeptidases cleave the C-terminal residues Aminopeptidases and carboxypeptidases are highly specific, thus are of limited use due to slow rates and resistance of some amino to cleavage III. Disulfide Bond Cleavage Disulfides are reduced to thiol with dithiothreitol (DTT) or 2- mercaptoethanol Thiols are treated with alkylating agents (e.g. iodoacetic acid) to prevent the re-oxidation during subsequent steps. 7
Protection of sulfyhydryl groups IV. Separation and Molecular Weight Determination of Subunits Traditional Methods SDS-PAGE, SEC, or RP-HPLC are used to separate the subunits after cleavage of disulfide bonds Mw standards and a calibration curve are used to determine the molecular weights The approximate number of amino acids can be estimated from the Mw of the subunit using 110 Da as the average molar mass for each amino acid Recent methods MALDI: more accurate and fast 8
V. Amino Acid composition Strategy: hydrolysis followed by separation and identification Acid catalyzed hydrolysis 6M HCl/ 100-120ºC/ 24 h (in oxygen free environment to prevent oxidation of SH groups) Some residues are degrated under these harsh conditions Base catalyzed hydrolysis 4 M NaOH /100ºC/ 4-8 hours Arg, Cys, Ser and Thr are decomposed and other amino acids are deaminated and racemized Used mainly to determine Trp which is extensively degraded under acid catalyzed hydrolysis V. Amino Acid composition Enzymatic hydrolysis By exo- and endopeptidases A combination of endo and exopeptidases must be used to hydrolyze all the peptide bonds Separation Individual amino acids in hydrolyzed mixture can be separated by RP-HPLC or CE and identified according to retention time Increasing sensitivity Pre- or post-column derivatization is used to increase sensitivity 9
Derivatization with OPA and MCE VI. Cleavage of Specific Peptide Bonds Direct sequencing is applicable to peptides that have up to about 50 residues only. Problems which occur after lengthy reactions Incomplete reactions Accumulation of impurities from side reactions Solution: use enzymes to break down the polypeptide chain into shorter fragments Proteolytic enzymes: endopeptidases and exopeptidases 10
Enzymatic Fragmentation Trypsin Trypsin is the most commonly used proteolytic enzyme. It cleaves at the C-end of positively charged amino acids (Arg and Lys) if the next residue is not a proline. It is highly specific Cleavage sites may be removed or added via derivatization to take advantage of the specificity of trypsin Reaction times can be adjusted to limit proteolysis if there are too many Arg and Lys residues Non-denaturing conditions can be used to limit proteolysis as well Trypsin Digestion 11
Derivatization of Cys for Tryptic Digestion Other Proteolytic Enzymes Endopeptidases Pepsin; cleaves at the amino end of Phe, Tyr, Trp the previous residue is not a proline Chymotrypsin: cleaves at the carboxyl end of Phe, Trp, Tyr if the next residue is not proline Endopeptidae GluC: cleaves at the carboxy end of Glu Exopeptidases Leucine aminopeptidase: cleaves rapidly N-terminal leucine aa. Does not cleave N-terminal proline Aminopeptidase M: cleaves all N-terminal residues Carboxypeptidase A: cleaves all except Arg, Lys, and Pro Especially efficient for aa with bulky aliphatic and aromatic side chains Does not cleave if the next residue is Pro Carboxypeptidase B: cleaves C-terminal Arg and Lys if the next residue is not Pro Carboxypeptidase C: cleaves C-terminal residues 12
Chemical Fragmentation Methods Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end forming a homoserine lactone 1. 3. 2. 4. Sequence Determination Separate segments by chromatography or electrophoresis and sequence fragments individually Edman degradation is the method of choice Fully automated systems which use the Edman degradation methods are available commercially (Sequenator) In the sequenator the protein is immobilized through bonding to a solid support or by adsorbing it onto an inert glass frit. Controlled amounts of reagents are injected by a pumping system The thiozolinone is transferred to a conversion chamber for hydrolysis to the PTH amino acid The final product, the PTH amino acid, is pumped into an HPLC column for on-line analysis 1 hour analysis time is possible for 50 amino acid residues 13
The solid-phase matrix-the Merrifield resin Edman degradation 14
Ordering of Peptide Fragments Compare amino acid sequence of one set of peptide fragments with the sequence of a second set of fragments obtained using different cleavage points Determination of Disulfide Bond Position Digest polypeptide chain(s) Run 2D gel of mixture of fragments using same conditions in both dimension After separation in the first dimension, the matrix is exposed to performic acid which cleaves all possible disulfide bonds Separation in the second dimension is performed Fragment without ss bonds will be positioned along the diagonal of the matrix Fragments linked by S-S bonds will produce off diagonal spots The disulfide linked fragments can be extracted from the gel and sequenced 15
Protein Sequencing by Mass Spectrometry Digest protein Obtain MALD TOF mass spectrum of digest Use online database to match fragments patterns with those in the data base Obtain sequence of fragments by performing MS/MS 16