BCHM 307 1st Edition Lecture 11 Outline of Last Lecture I. Protein AnalysisA. Two-dimensional Gel ElectrophoresisB. Definition of Isoelectric PointII. Protein CleavageA. Definition of ProteasesB. Examples of ProteasesIII. Protein SequencesOutline of Current Lecture I. Mass SpectrometryA. Definition of Ionization SourceB. Definition of Mass AnalyzerC. Definition of DetectorII. In-Gel Protease DigestionIII. Unknown Protein AnalysisA. Peptide Mass FingerprintingB. Tandem Mass SpectrometryIV. EnzymesA. Definition of EnzymeB. Definition of SubstrateC. The Two Enzyme ModelsCurrent LectureThe process of mass spectrometry revolutionized biochemistry. This process refers to measuring the mass of molecules very precisely. The instrument that performs this task is calleda mass spectrometer. As mentioned last lecture, proteins can be cut up into peptide sequences using proteases. These peptides are put into a solvent.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.The first step of mass spectrometry is to put this mixture into an ionization source. An ionization source adds a charge to the peptides and turns them into their gas form. This is due to the fact that only charged molecules in the gas phase can be analyzed with a mass spectrometer. The charged peptides then move to the mass analyzer. The mass analyzer separates the ions based on mass and charges using a vacuum and electromagnetic field. The molecules go into a detector. The detector is a device that turns the arrival of the ions into computer-readable electrical signals. The computer processes the signals into a graph with a series of peaks.In-gel proteases digestion is another analytical technique used. This a way to prepare peptide samples to be analyzed by mass spectrometry. The SDS-PAGE technique is used and then a certain protein band is cut out of the gel. This is used if you are interested in a single band. Normally trypsin is used as the protease. An organic solvent can be used to extract the peptides and then put through mass spectrometry.If you have a protein whose identity is unknown, peptide mass fingerprinting (PMF) can be used. It is based on the concept that each protein has its own unique sequence of peptides after performing a site-specific proteolysis. The masses of known peptides can be used to identify what the whole protein is. There are three requirements in order to do this process. 1. The organism be studied must have had its genome sequenced. 2. A site-specific protease must be used to break up the protein into peptides. 3. A high accuracy mass spectrometer must be used. The computer will create a theoretical graph that can be compared to experimental peptide masses. A list of possible proteins is then generated. Tandem mass spectrometry can also be used to identify a protein. This process focuses on the complete amino acid sequence. It is based on the principle that almost every amino acid has its own unique residue mass, which it contributes to the peptide formation. Scientists know how peptides tend to break apart, based on these residual masses. The fragments produced canbe analyzed to determine what the original protein sequence was.Now we are moving onto the chapter about enzymes. An enzyme is a catalyst, often a protein, which increases the rate of a reaction without being consumed. The most efficient enzymes enhance the rate of a reaction by 1020 fold. Enzymes are often recognized by their names ending with the suffix –ase, though this is not always the case as with Trypsin or Chymotrypsin. Enzymes increase the rates of reactions that were originally possible, but slow. They decrease the activation energy of a reaction in order to do this. Enzymes do not alter the amount of energy used by the reaction, known as standard free energy change. They also do not affect the equilibrium constant of a reaction. Enzymes are also highly selective.A substrate is anything that interacts with an enzyme. There are two models that show how enzymes interact with their substrates. The lock and key model shows that the substrate “key” fits into an enzyme “lock” active site shaped specifically for that key. The other is called the induced fit model. This model says that the substrate will bind to an area on the enzyme that isn’t the active site. This binding will cause the enzyme to change its shape in order to fit around the substrate, leading to an exact fit. In both cases once catalysis has taken place the substrate will no longer fit in the enzyme and will be
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