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University of Toronto Scarborough
Biological Sciences
Shelley A.Brunt

lec04 lecture outline 1. analytical techniques 2. protein sequencing 3. protein structure analytical techniques 1. electrophoresis a. separates proteins based on migration in an electric field based on i. size ii. charge b. matrix usually polyacrylamide i. AKA PAGE 1. polyacrylamide gel electrophoresis c. type of electrophoresis based on size only i. SDS (sodium dodecyl sulfate) PAGE 1. all proteins are coated with SDS (detergent) giving all proteins a net negative charge a. binds to the hydrophobic regions of proteins 2. because of the way SDS binds (one molecule per every two residues) it has the same charge to mass ratio a. anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to it 3. requires a reducing agent to break disulfide bridges a. reducing agents i. beta mercaptoethanol ii. dithiothreitol how do you produce a protein population which has the same charge to mass ratio so that separation is based on molecular weight with charge having little to no influence? 1. use a detergent (SDS) 2. binds to proteins in a way that results in constant charges across all proteins (negative  anode) a. isolate proteins for size discrimination i. place in PAGE to separate proteins by size only SDS PAGE 1. analytical 2. can be used to purify proteins for mass spec / sequencing 3. proteins with decreasing molecular weight settle at the bottom a. because smaller molecules are able to penetrate the pores of the gel beads, and thus progress more slowly than larger molecules 4. proteins negatively charged move towards the anode (positively charged electrode) 5. example of one dimensional separation 6. slide 8 a. protein present in each of the lanes b. it is more intense in relation to other proteins at 40,000 MW c. procedure to obtain desired protein i. purification by 1. gel permeation 2. ion exchange column ii. analyze the fractions that come off with SDS PAGE iii. safe to ignore proteins at undesired molecular weights IEF 1. isoelectric focusing a. two dimensional gel electrophoresis i. combines 1. isoelectric focusing in the first dimension a. pI b. charge separation 2. SDS PAGE in the second dimension a. size 3. isoelectric focusing in first dimension  bands run into gel and separate in SDS-PAGE in the second dimension ii. function 1. used to identify proteins which change in expression 2. useful in proteomics a. mass spec b. peptide cleavage c. requires larger gels to separate two dimensions i. IEF ii. SDS PAGE 2. genomics a. looking at DNA and RNA b. but not all answers lie in DNA and RNA c. look at proteins  proteomics i. role of methylation in DNA 1. how it controls expression in DNA a. concept of epigenetics ii. proteomics 1. study of proteins a. isolates proteins with specific pI and molecular weight i. each spot on the IEF represents an isolated protein 1. may be isoforms of the desired protein 2. not necessarily completely different a. differ in terms of post translational modification from other organisms identifying proteins 1. key players a. electrospray mass spectrometry b. matrix assisted desorption ionization (MALDI) 2. mass spectrometry a. determines mass of a molecule b. at most basic level uses the time it takes for a charged gas phase molecule to travel from the point of injection to a detector c. the time depends on the charge and mass of the molecule i. data is reported as a mass to charge ratio d. limited in protein identification because proteins were too large i. need to analyze protein molecules rather than just amino acids 3. electrospray mass spectrometry a. pumps proteins through a metal needle at high voltage to create droplets i. charged proteins become focused on the detector 4. matrix assisted desorption ionization (MALDI) a. ionization technique used in mass spec i. allows analysis of biomolecules which tend to be fragile and fragment when ionized by more conventional ionization methods b. MALDI is a two step process i. desorption triggered by UV laser beam 1. matrix material absorbs light 2. leads to ablation of upper layer of UV laser light a. creates many species of i. neutral and ionized matrix molecules ii. protonated and deprotonated ii. analyte molecules are ionized c. proteins are mixed with a chemical matrix and precipitated on a metal substrate i. chemical matrix absorbs light at a particular wavelength ii. a laser at the proper wavelength imparts energy to the protein molecule 1. causes protein to move iii. protein is released (desorbed) and is directed to the detector (time of flight - TOF) 1. MALDI-TOF a. measures mass to charge ratio b. not proteins  peptides c. generates graph / peptide profile i. height of peak represents amount of peptide ii. length measures mass to charge ratio d. similar protein  similar peptide profile  suggests it is same protein i. comparison via database of protein profiles iv. can be used to also determine protein sequence v. usually requires protein cleavage into shorter amino acid chains 1. use of proteases amino acid composition 1. in the average protein  typical types of distribution a. tend to see aspartates and glutamates b. cysteine and tryptophan relatively uncommon i. cysteine used in active sites and forming disulfide bonds 2. to determine amino acid composition  break peptide bonds a. using acid hydrolysis and high performance liquid chromatography (HPLC) b. using peptide cleavage and mass spec acid hydrolysis and HPLC 1. break peptide bonds via acid hydrolysis to form respective amino acids 2. treat amino acid hydrolysate (substance formed by hydrolysis) with phenylisothiocyanate (PITC) a. so that it can be identified by HPLC b. becomes PTC-amino acid i. phenylthiocarbamoyl-amino acid 3. put it through HPLC to analyze chromatograph from HPLC 1. separation of the PTC amino acids in the column a. represents amino acids present in original protein b. amount is equal to the area under the respective peak 2. limitations a. some codes not recognized as amino acid codes i. why b. during treatment with PITC  amino acid gets converted into amide 3. advantages a. gives an idea of composition primary structure of protein 1. different proteins have different numbers of subunits and associated molecular weights 2. some proteins are made up of a lot of subunits 3. amino acid encodes proteins  but may not be a functional protein  it is actually a polypeptide (which may or may not function by itself) 4. homo or hetero  same or different subunit polypeptide comes together to form functional protein protein sequence determination 1. protein sequence or composition 2. protein sequence  need to reduce disulfide bonds first 3. composition  enzymatically break into smaller pieces  determine the sequence of each peptide 4. to determine sequence  need more than one chemical to allow cleavage and reformation of protein sequence 5. protein sequence determination a. e.g. protein with two different polypeptide chains linked by disulfide bonds i. reduce disulfide bonds to separate chains 1. use chemical or enzymatic methods to break each polypeptide into smaller peptides a. determine the sequence of each peptide fragment 2. use different methods to generate a different set of peptide fragments a. determine the sequence of each peptide fragment ii. use the two sets of overlapping peptide sequences to reconstruct the sequence of each poly peptide 1. how are they overlapping? 2. how do you reconstruct the polypeptide? iii. repeat fragmentation without breaking disulfide bonds to identify the cys- containing sequences involved in the disulfide linkages sequencing amino acids 1. two ways to sequence amino acids a. edman degradation procedure i. old way of sequencing ii. expensive iii. manual method b. tandem mass spec i. less expensive ii. automatic edman degradation procedure 2. allows identification of one residue at a time from the n-terminus a. thus protein sequencing 3. treat protein at pH 9.0 with PITC a. aka edman reagent 4. PITC reacts with the free n-terminal residue of the amino acid chain to form a PTC-peptide 5. treat PTC peptide with anhydrous acid (TFC - trifluoroacetic acid) a. the peptide bond is selectively cleaved leading to anilinothiazolinone derivative of the residue i. each time you remove an amino acid, you get a new n-terminus 1. repeat cycle until all amino acids identified a. usually lasts 20-30 amino acids until fail i. each round has a different amino acid terminal end 1. sequence one by one with PITC for entire peptide a. or until fail 6. this method is stable for identification by chromatography (HPLC) a. have to remove disulfide bond whenever it is present i. therefore need to reduce disulfide bond  stabilize reduced structure 1. only stable reduced derivative can be identified via HPLC ii. removal of disulfide bonds to allow release of cysteine during edman degradation 1. cystine  2 cysteine residues 2. treat reduced cysteine with alkylating agent (iodoacetate) which converts the free cysteine to stable (S) - carboxymethylcysteine a. no reformation of disulfide bonds in presence of oxygen b. stable residue prevents reformation of disulfide bonds tandem mass spectrometry for peptide sequencing 1. susceptible to n-terminal blockage a. edman does not deal with post-translationally modified proteins b. mass spec does 2. tandem mass spec a. involves multiple steps of mass spec selection with some form of fragmentation occurring in between the stages i. fragmenting the sample inside and analysing the products generated ii. used for structural and sequencing studies b. take generated peptides i. size them ii. whether or not you can identify peptide depends on database profiles 1. if not, then resequence to verify a. with what? use of cleavage agents and proteases 1. to generate short amino acid chains for use in edman or mass spec for sequencing a. proteins are too long to allow complete analysis without first selectively cleaving at some peptide bonds i. anything larger than 40 or shorter than 100 amino acids 2. questions to ask a. how chemical agents work to create short peptides b. role of proteases to cleave peptides c. presence of proteases hinder identification of proteins i. how? 3. peptides desired at longer than 40 to less than 100 amino acids 4. what can you use? a. chemicals b. endopeptidases i. cleaves at the inside of the protein (middle) c. exopeptidases i. cleaves at the outside of the protein (end terminals) protein cleavage with chemicals 1. cyanogen bromide a. used to reduce the residues to allow for edman degradation b. selective process c. cleavage on c-terminal side of methionine 2. methionines and protein sequences a. not common i. biological reason
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