Introduction to Proteomics

Introduction to Proteomics
Slide 1: Introduction to Proteomics

Outline What is proteomics? Why study proteins?
Discuss proteomic tools and methods Slide 2: Outline Voice: The purpose of this presentation is to introduce proteomics. We will begin by answering two questions What is proteomics and Why study proteins. Then we will discuss proteomic tools and methods. So let’s begin!

What is proteomics? Slide 3: What is proteomics?
Voice: The first obvious question is “What is proteomics”

Proteomics is the analysis of the protein complement to the genome
Genomics Proteomics Gene Transcript Protein Slide 4: Proteomics is the analysis of the protein complement to the genome. Voice: The answer, proteomics is the analysis of the protein complement to the genome. Simply put as genomics is the study of all gene, the study of all proteins is proteomics. It is the study of all the proteins in a cell, tissue, organ, leaf, etc. Basically, proteomics involves the analysis and identification of proteins.

…while the genome is a rather constant entity, the
proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle and in different environmental conditions.” “..the large-scale study of proteins…while it is often viewed as the “next step”, proteomics is much more complicated than genomics. Slide 5: Quotes Voice: That’s the simple definition of proteomics. A more extensive description of proteomics is...One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle and in different environmental conditions.” …while the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. “..the large-scale study of proteins…while it is often viewed as the “next step”, proteomics is much more complicated than genomics Wikipedia,

Proteomics is multidisciplinary
Protein Biochemistry Biology Analytical Chemistry Proteomics Bioinformatics Molecular Biology Slide 6: Proteomics is multidisciplinary Voice: Proteomics includes many traditional scientific disciplines including, biology, protein biochemisty, analytical chemistry, and other newer disciplines such as bioinformatics and molecular biology.

Proteomics Research Basic research:
To understand the molecular mechanisms underlying life. Applied research: Clinical testing for proteins associated with pathological states (e.g. cancer). Slide 7: Proteomics Research Voice: We uses these combined disciplines to conduct proteomic research. There are two categories of research. In basic research, we use proteomics to understand the molecular mechanisms of life. These question include What is my protein of interest and how abundant is it? In applied research, we may use clinical testing for diseases associated with our protein of interest.

Applications of Proteomics
Glycoyslation Phosphorylation Proteolysis Yeast two-hybrid Co-precipitation Phage Display Drug Discovery Target ID Differential Display Yeast Genomics Affinity Purified Protein Complexes Mouse Knockouts Medical Microbiology Signal Transduction Disease Mechanisms Organelle Composition Subproteome Isolation Protein Complexes Structural Proteomics Proteome Mining Post-translational Modifications Protein Expression Profiling Functional Proteomics Protein-protein Interactions Proteomics Slide 8: Applications of Proteomics Voice: Both methods of research may have many applications. For example, drug discovery, mice knockout that glow in the dark and even disease mechanisms

For example: Hemoglobin
Picks up oxygen in the lungs, travels through the blood, and delivers it to the cells. O2 hemoglobin Hbβ Hbα Slide 9: For example: Hemoglobin Voice: For years we have carefully study a protein called hemoglobin, which picks up oxygen in the lungs, travels through the blood, and delivers it to cells. It have for subunits two are alpha and two are beta.

Sickle cell disease is caused by a single amino acid change.
Normal Hbβ Mutated Hbβ ATG GTG CAC CTG ACT CCT GAG GAG … ATG GTG CAC CTG ACT CCT GTG GAG … E M V H L T P … Slide 10: Sickle cell disease is caused by a single amino acid change. Voice: But when a mutation arises, a deadly disease occurs. In the HBb, a single nucleotide may changes from adenine to thymine causing a change from a glutamine to a valine in the protein sequence. The mutated HbB becomes sticky and aggregation to produce sickle shaped red blood cells. These sickled shaped blood cells are flexible and sticky. They block capillaries and blood flow to organs. The blockages may result in pain, infection, and/or organ damage. 1 out of every 500 people of african descent have this disease. We can use proteomics to identify the fatal mutations along with their disease. Hopefully we can take it much further and also use proteomic clinical application to generate treatments for diseases.

Summary – what is proteomics?
Involves the study of proteins Proteomics is multidisciplinary Proteomics is being applied to both basic and clinical research Slide 11: Summary – what is proteomics? Voice:

Why study proteins? Slide 12: Why study proteins?
Voice: Now that we know proteomics is the study of the proteome another question arises. Why exactly should we study proteins?

What are PROTEINS? Proteins are large, complex molecules that serve diverse functional and structural roles within cells. Slide 13: What are PROTEINS? Voice: We know from our everyday lives that proteins are necessary component of our diet. We need proteins to keep our immune systems healthy, grow fabulous hair, bulging muscles, and much more. Great source of protein include beans, meat, and diary products. But exactly what are proteins? Proteins are large biological molecules that serve diverse functional and structural roles within the cell.

Proteins do most of the work in the cell
Enzyme Protease Degrades Protein O 2 Transport Hemoglobin Carries O2 Motion Actin Contracts Muscles Regulation Insulin Controls Blood Glucose Slide 14: Proteins do most of the work in the cell Voice: Proteins can be classified according to a wide range of functions in the cell. For examples, antibodies act in defense of the cell. They bind to specific foreign molecules, such as viruses and bacteria, to help protect the cell. Enzymes perform thousands of chemical reactions. Regulator proteins control biological processes within cells, tissues, and organs. Many proteins, like keratin and actin, provide structure, support, and range of motion. Transport and storage proteins, like hemoglobin, bind and carry atoms and small molecules within cells and throughout the body. Support Keratin Forms Hair and Nails Defense Antibody Fights Viruses

Proteins are comprised of amino acid building blocks
H H2 R2 H C N O C OH + R C H Variable H2O N H Slide 15: Proteins are comprised of amino acid building blocks Voice: Each amino acid contains a R group, a carboxyl group, an amino group and a hydrogen all bound to a central carbon atom. To form a covalent link between amino acids, the carboxyl group of amino acid 1 and the amino group of amino acid 2 must undergo dehydration synthesis. Dehydration synthesis links the amino acids and produces a molecule of water. The covalent link between amino acids is called a peptide bond. R1 O C R2 H N H2 H Base Dipeptide Peptide Bond

Each amino acid has unique chemical properties.
basic Histidine Lysine Arginine Aspartate Glutamate acidic non-polar hydrophobic Valine Phenylalanine Proline Methionine Tryptophan Isoleucine Leucine Alanine Slide 16: Each amino acid has unique chemical properties. Voice: Each amino acid has a R group. The R group provides the amino acid with unique chemical properties. 20 amino acids in nature - structurally and functional diverse. These 20 amino acid belong to 4 different classes: basic, acidic, non-polar hydrophobic, and polar hydrophilic. polar hydrophilic Glutamine Tyrosine Serine Cysteine Asparagine Threonine Glycine

Proteins are chains of amino acids.
OH N H Short chains of amino acids are called peptides. Slide 17: Proteins are chains of amino acids. Voice: Proteins are large molecules made up of several building blocks called amino acids. Short chains of amino acids are called peptides. Proteins are made of many peptide subunits and are often called polypeptide molecules. Proteins are polymers of amino acids joined by amide bonds N H Proteins are polypeptide molecules that contain many peptide subunits.

Messenger Ribonucleic Acid (mRNA) Amino Acid-transfer RNA
Gene Nucleus 3’ tRNA Trp Messenger Ribonucleic Acid (mRNA) tRNA Ala Met tRNA Met Ala Met Ala Trp Amino Acid-transfer RNA Large Subunit Ribosome 5’ Empty tRNA Empty tRNA Met Small Subunit A U G G C C U G G U A G Ribonucleotides A U G C Cytoplasm Slide 18: Translation is the synthesis of proteins in the cell. Voice: In the cell, proteins are synthesized during a process called translation. There are 3 major steps in translation. In step 1, the small ribosomal subunit binds the mRNA sequence and is joined by the large subunit. After ribosome formation, the initiator transfer RNA or tRNA is bound to the amino acid Methionine. The codon AUG codes for the Methionine amino acid tRNA complex. This complex binds to the first site within the ribosome. In the elongation step, tRNA with bound amino acids, here Alanine, binds with next codon. The Methionine is covalently linked to Alanine by a peptide bond. Then the ribosome moves one codon downstream and the initiator tRNA is released. The elongation step continues and Tryptophan is added to the amino acid chain. Once the elongation step is completed, the termination step begins. In the termination step, the ribosome reaches a stop codon which does not encode a tRNA. During this step the protein chain is released. Codon 1 A U G = Methionine U G Codon 3 Tryptophan = C G Codon 2 = Alanine U G Codon 4 Stop = A Translation is the synthesis of proteins in the cell.

Proteins have specific architecture
Slide 19: Proteins have specific architecture Voice: We now have a complete amino acid chain. But how does it fold?

Proteins arrive at their final structure in an ordered fashion
Slide 20: Proteins arrive at their final structure in an ordered fashion Proteins consist of a long amino acid chain folded into complex shapes. There have 4 basic levels of protein structure: primary, secondary, tertiary, and quaternary. The primary structure is the linear chain of amino acids. Bonds may form between nearby amino acids within the primary structure. The bonds may produce folds and coils. These coils are called alpha helices and the folds are called beta-pleated sheet. The secondary structure is primarily composed of alpha helices and beta-pleated sheets. The alpha helices and beta-pleated sheets fold together to form a 3D shape or the tertiary structure. Many tertiary structures or folded subunits may assemble into a single cluster. These clusters are called quaternary structure. It is important to remember that protein must fold properly in order to perform their functions. Improperly folded proteins may cause serious slow downs or ceases of functions. These alterations in function may even cause diseases such as Alzheimers or Mad Cow Disease. J. E. Wampler, 1996,

Summary – why study proteins?
Biological workhorses that carry out most of the functions within the cell Serve diverse functional and structural roles Composed of amino acids that are covalently linked by peptide bonds Synthesized during the translation process Must fold correctly to perform their functions Slide 21: Summary – why study proteins? Proteins are large biological molecules that serve diverse functional and structural roles in the cell. They are biological workhorses and carry out most of the cellular functions. Proteins are synthesized during translation where amino acids are linked by peptide bonds. However, proteins much form their correct structures in order to function properly.

Proteomic tools and methods
Slide 22: Tools to study proteins Voice: Now that we know what proteomics is and how important it is to study protein. We will discuss the proteomic tools that I use every day to study proteins.

Proteomic tools to study proteins
Protein isolation Protein separation Protein identification Slide 23: Tools to study proteins Voice: Proteomic tools can be divided into three categories. Protein isolation, protein separation, and protein identification.

Protein Isolation Slide 24: Protein Isolation
Voice: In order to study proteins, we have to extract them from the sample, whether its blood, leaves, or bacteria.

How are proteins isolated?
Mechanical Methods grinding – break open cell centrifugation – remove insoluble debris Chemical Methods detergent – breaks open cell compartments reducing agent – breaks specific protein bonds heat – break peptide bonds to “linearize” protein Slide 25: How are proteins isolated? Voice: To isolate protein we use both mechanical and chemical methods. We use mechanical methods like grinding and centrifugations for opening cells and removing debris. We also use chemical reagents like detergents, reducing agents, and heat for opening cell compartments, and breaking peptide bonds.

Protein isolation procedure
Grind sample in buffer Find a sample Pick it Transfer to tube Slide 26: Protein isolation procedure Voice: In any real study, we start with a biological sample: a piece of tissue, a plate cultured cells, a flask of bacteria, a leaf, and so on. Grind samples in a buffer that contains many of the chemical reagents we discussed on the previous slide. After grinding, the liquid or supernatant is transferred to a new tube. The tube is heated to denature or linearize the protein. The tube is centrifuges the pellet and remove any insoluble debris. Again, the liquid phase or supernatant is transferred to a new tube. This final tube contains the pure protein solution. But what is that floating in our tube? Centrifuge to remove insoluble material Heat the sample “pure” protein solution Recover supernatant Keep solution for gel analysis

Protein X “pure” protein solution Isolated Protein X
Slide 27: Protein X Voice: It’s protein X. For the purpose of learning about proteomics, we will identify protein x using the proteomic tools and methods. We will continue to analyze protein x throughout the remainder of the presentation.

Summary – protein isolation
Proteins can be isolated from a variety of samples Proteomics includes the use of both mechanical and chemical methods to isolate proteins Opening cell or cellular compartments Breaking bonds and “linearizing” proteins Removal cell debris Slide 28: Summary – protein isolation Voice:

Protein Separation SDS-PAGE Slide 29: Protein Separation
Voice: Now that we have isolated protein, we need to separate the proteins.

“PURE” Protein Solution
Why separate proteins? “PURE” Protein Solution Tube 1 Decreased Protein ID Increased Complexity Tube 2 Increased Protein ID Decreased Complexity Slide 30: Why separate proteins? Voice: But why? We separate protein to increase our chance of identifying proteins. For example here we have our pure protein solution that includes protein x shown in white. The sample has many proteins or increased complexity. Because of all of the blue proteins, our chances of identifying protein x have low. But if we reduce complexity by removing blue proteins, as you see in tube 2, we increase our chances of identifying protein x.

How to separate proteins?
Separating intact proteins is to take advantage of their diversity in physical properties, especially isoelectric point and molecular weight Slide 31: How to separate proteins? Voice: To separate intact proteins, we take advantage of their physical properties, especially, the isoelectric point and molecular weight.

Methods of Protein Separation
Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE) Isoelectric Focusing (IEF) Slide 32: Methods of Protein Separation Voice: There are 2 major methods for proteins separation: sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF). Today we will discuss both of these.

SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a widely used technique to separate proteins in solution Slide 33: SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to separate proteins in solution Voice: So let’s begin. What is SDS-PAGE? SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to separate proteins in solution only by the molecular weight.

SDS-PAGE separates only by molecular weight
Molecular weight is mass one molecule Dalton (Da) is a small unit of mass used to express atomic and molecular masses. Slide 34: SDS-PAGE separates only by molecular weight Voice: Molecular weight is the mass of one molecule. Molecular weight is often expressed in daltons. Dalton (Da), is a small unit of mass used to express atomic and molecular masses. It is defined to be one twelfth of the mass of an unbound atom of carbon-12.

PAGE is widely used in Proteomics Biochemistry Forensics Genetics
Molecular biology Slide 34: PAGE is widely used in Voice: PAGE is widely used in biochemistry, forensics, genetics, molecular biology and of course proteomics.

Polyacrylamide gels separate proteins and small pieces of DNA
Major components of polyacrylamide gels Acrylamide – matrix material/ NEUROTOXIN Bis-acrylamide - cross-linking agent/ NEUROTOXINS TEMED - catalyst Ammonium persulfate - free radical initiator Slide 35: Polyacrylamide Gels separate proteins and small pieces of DNA Voice: Polyacrylamide Gels separate proteins and small pieces of DNA. They are composed principally of: acrylamide the matrix material, bis-acrylamide the cross-linking agent, TEMED a catalyst of the free radical initiator ammonium persulfate. But how does it work?

Polyacrylamide (non-toxic)
H 2 O A c r y l a m i d e (matrix material) H N O B i s a c r y l m d e (cross-linking agent) Polymerization N S O 4 T E M D (catalyst) A m o n i u p e r s l f a t (free radical initiator) Polyacrylamide (non-toxic) Slide 36: One-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) Voice: To make a 2D gel, we first pour liquid acrylamide in the flask. Then we add the powder bis acrylamide and shake. Next we add the free radicals and catalyst and shake. We pour our liquid mix into a cast. Over time the gel will polymerize or become a solid. Finally we have a polyacrylamid gel.

Polyacrylamide (non-toxic)
H C 2 Bis-acrylamide cross links Polyacrylamide (non-toxic) Slide 37: One-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) Voice: The polyacrylamide gel is composed of the acrylamide matrix cross linked with bis-acrylamide.

Sodium dodecyl sulfate - SDS
The anionic detergent SDS unfolds or denatures proteins Uniform linear shape Uniform charge/mass ratio Slide 38: Sodium dodecyl sulfate – SDS Voice: SDS is also added to many polyacrylaminde gels. When SDS is added, it binds to the protein. When SDS binds it does two things 1) it neutralize the protein and imparts are negative charge and 2) it denatures or linearizes the protein.

One-dimensional polyacrylamide gel electrophoresis (SDS-PAGE)
Cathode (-) Slide 41: One-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) Voice: The protein samples are applied to one to each lane of the polyacrylamide gels in a buffer. An electric current is applied across the gel, causing the negatively-charged proteins to migrate through the gel towards the anode. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty because they encounter more resistance. Anode (+) Standard Sample1 Sample2

During SDS-PAGE proteins separate according to their molecular weight
Cathode (-) 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa Slide 40: Visualize bands by staining with Coomassie Brilliant Blue Voice: After a set amount of time, usually a few hours, the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin. Therefore, proteins may be separated roughly according to size and therefore, molecular weight. Following electrophoresis, the gel may be stained, allowing visualisation of the separated proteins, or processed further. After staining, different proteins will appear as distinct bands within the gel. It is common to run molecular markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker. 25 kDa 20 kDa Bromophenol Blue dye front Anode (+) Standard Sample1 Sample2

Image of Real SDS-PAG Cathode 250 kiloDaltons 150 kDa 100 kDa 75 kDa
Slide 41: Coomassie-stained SDS-PAGE gel Voice: This is the image of a real SDS-PAG. You can see many band in the gel. 37 kDa 25 kDa 20 kDa Anode

Separation of Protein X
Cathode (-) 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa Slide 42: Visualize bands by staining with Coomassie Brilliant Blue Voice: In our separation of the protein by SDS-PAGE, we see protein x. The SDS-PAG shows us that protein X is about 25 kDa. However, in the analysis we see that all the protein are not separated. There may be other protein in that 25 kDa band along with Protein X. So how can we see only Protein X 25 kDa Protein X 25 kDa 20 kDa 11 kDa Bromophenol Blue dye front Anode (+) Standard Sample1 Sample2

Two-dimensional gel electrophoresis (2-DGE)
1st dimension - isoelectric focusing 2nd dimension - SDS-PAGE Most widely used protein separation technique in proteomics Capable of resolving thousands of proteins from a complex sample (i.e. blood, organs, tissue…) Slide 43: Two-dimensional gel electrophoresis Voice: To separate protein x, we can use two-dimensional gel electrophoresis . During 2-DGE we use isoelectric focusing in the first dimension and SDS—PAGE in the 2nd dimension. 2DGE is the most widely used separation technique in proteomics and it is capable of resolving thousands of proteins.

1st Dimension-Isoelectric Focusing
Isoelectric focusing (IEF) is separation of proteins according to native charge. Slide 44: Two-dimensional gel electrophoresis Isoelectric focusing is separation of proteins according to native charge. Voice: The first separation in 2DGE is called isoelectric focusing or IEF. During IEF, proteins are separated on a pH gradient. Protein will migrate to the point in the gradient were their net charge equals zero or is neutral. That point is called the isoelectric point. For example a protein maybe +2 charge at pH 5 but at pH8 it is neutral. This is the isoelectric point. isoelectric point -pH at which net charge is zero

2-DGE protein samples 1st dimension IEF 2nd dimension SDS-PAGE
pH gradient 10 3 IEF 1st dimension Neutral at pH 3 SDS-PAGE 2nd dimension 20 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 150 kDa 11 kDa Slide 45: Two-dimensional gel electrophoresis Voice: In 2DGE, the purified proteins are subsequently applied to a pH gradient strip submerged in a suitable buffer. An electric current is applied across the strip, causing the charged proteins to migrate across the gel toward their isoelectric point. For example, this band represents proteins with a pI of 3. The pH gradient strip is placed onto of a SDS-PAG. Proteins from the pH gradient strip migrate into the gel. Depending on their size, each protein will move differently through the gel matrix. Following electrophoresis, the gel may be stained, allowing visualisation of the separated proteins, or processed further. After staining, different proteins will appear as distinct spots within the gel. It is common to run molecular markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

2-DG pI kDa 3 4 5 6 7 8 9 10 100 75 mass 50 Slide 46: Two-dimensional gel electrophoresis Voice: This is the image of a real 2D SDS-PAG. You can see hundreds and hundreds of spots on the gel. 25 Arabidopsis developing leaf

2-DGE 2nd dimension SDS-PAGE Protein X 25 kDa pI 5 3 4 5 6 7 8 9 10
Slide 47: Two-dimensional gel electrophoresis Voice: But what about protein x? We can see protein x on our 2DG. We see protein x is still about 25 kDa. We also see now that protein x has a isoelectric point at 5. 11 kDa Protein X 25 kDa pI 5

1-DGE vs. 2-DGE 1-DGE (SDS-PAGE) 2-DGE High reproduciblity Quick/Easy
Separates solely based on size Modest resolution, dependent on complexity of sample Modest reproducibility Slow/Demanding Separates based on pI and size High resolution, not dependent on complexity of sample Slide 48: 1-DGE vs. 2-DGE Voice: SDS-PAGE has several great advantages, whether used alone or in combination with isoelectric focusing. When used alone, SDS-PAGE or 1DGE, its reliability, reproducibility and ease make is a common choice to separate proteins only by their molecular weigh. 2-DGE combines isoeletric focusing (IEF) and SDS-PAGE to separate proteins by isoelectric point and molecular weight; but with modest reproducibility.

Summary – protein separation
Protein separation takes advantage physical properties such as isoelectric point and molecular weight SDS-PAGE is a widely used technique to separate proteins 1-DGE is a quick and easy method to separate protein by size only 2-DGE combines isoeletric focusing (IEF) and SDS-PAGE to separate proteins by pI and size Slide 49: Summary – protein separation Voice: Protein separation takes advantage of protein diversity including physical properties such as isoelectric point and molecular weight. SDS-PAGE is a widely used technique to separate proteins. 1-DGE is a quick and easy method to separate protein by size only but it has modest protein resolution that is sample dependent. 2-DGE combines isoeletric focusing (IEF) and SDS-PAGE to separate proteins by pI and size; but the process can be technically demanding.

Protein identification
mass spectrometry Slide 50 Protein identification Voice: The size and pI of a protein are not enough information to identify it. So, how do we tackle the protein identification problem?

Peptide mass fingerprinting
intact protein x protein digestion Make proteolytic peptide fragments - Digest the protein into peptides (using trypsin) mass spectrometry m/z intensity Measure peptide masses - “Weigh” the peptides in a mass spectrometer mass Slide 51: Peptide mass fingerprinting Voice: We will use a protein identification technique called peptide mass fingerprinting. Peptide mass fingerprinting is a protein identification technique in which MS is used to measure the masses of peptide fragments. The protein then is identified by matching the measured peptide masses to corresponding peptide masses from protein database. First, we make peptide fragments from an intact protein. To do this we digest the intact protein, in this case protein x, into peptides. Next we, use mass spectrometry to measure or weigh the peptide masses. Then finally, we match those peptide masses to peptides of known proteins. Match peptide masses to protein or nucleotide sequence database - Compare the data to known proteins and look for a match Protein ID

Protein digestion We use the enzyme TRYPSIN to digest (cut) proteins into peptides – trypsin cuts after Lysine (K) and Arginine (R) Protein X Slide 52: Protein identification Voice: In peptide mass fingerprinting, we break down or cut the proteins in to little pieces. This is called a protein digestion. To do this, we use a protease, like trypsin. We use the enzyme TRYPSIN to digest (cut) proteins into peptides – trypsin cuts after Lysine, K and Arginine, R. This is unknown sequence of protein x. We will cut this sequence with trypsin and as you see we get 3 peptides.

How does mass spectrometry identify unknown proteins?
Slide 53: How does mass spectrometry assist proteomics research? Voice: Now that we have our unkown peptides, how can we measure them.

Basics of mass spectrometry
determination of mass to charge ratio (m/z) Mass spectrometer = very accurate weighing scales third or fourth decimal place Slide 54: Basics of mass spectrometry Voice: A great method to measure the mass of peptides is through mass spectrometry. The goal of mass spectrometry is to determine the mass to charge ration of peptides. Mass spectrometers are very accurate weighing tools. They can be accurate to even the third or fourth decimal place.

We then “weigh” these peptides with a Mass Spectrometer
????????K ?????R ???????? Slide 55: We then “weigh” these peptides with a Mass Spectrometer Voice: So we are going to use mass spectrometry identify our unknown peptides and ultimately protein x. Mass Spectrometer

We then “weigh” these peptides with a Mass Spectrometer
????????K ?????R ???????? Da Da Da Slide 56: We then “weigh” these peptides with a Mass Spectrometer Voice: The unknown peptides are analyzed by mass spectrometry. The mass spectrometer produces the mass of each unknown peptide.

Mass of peptides should be compared to theoretical masses of known peptides
? ????????K = Da ?????R = Da Slide 57: Mass of peptides should be compared to theoretical masses of known peptides Voice: The mass of each unknown peptide is put into a computer. The computer compares the mass of the unknown peptides to a list of known peptides. ???????? = Da

Computation of theoretical masses of known peptides known
Computer Peptides WEGETMILK ADEMTYEK PLMEHGAK LMEHHH ASTEER DMGEYIILES EGEDMPAFY CYHGMEI EFPKLYSEK YSEPYSSIIR IESPLMIA AEFLYSR DLMILIYR METHIPEEK KISSMER PEPTIDEK MANYCQWS TYSMEDGHK YMEPSATFGHR GHLMEDFSAC HHFAASTR ALPMESS Proteome = all protein sequences Slide 58: Computation of theoretical masses of known peptides known Voice: The known peptides are generated by a computer. We build protein databases and put in the computer. Then the computer digest the protein database with “simulated trypsin”. This digestion produces a list of known computer peptides along with their estimated masses. Digest Proteome with simulated Trypsin

Mass of peptides compared to theoretical masses of all peptides known, using a computer program.
Computer Peptides WEGETMILK ADEMTYEK PLMEHGAK LMEHHH ASTEER DMGEYIILES EGEDMPAFY CYHGMEI EFPKLYSEK YSEPYSSIIR IESPLMIA AEFLYSR DLMILIYR METHIPEEK KISSMER PEPTIDEK MANYCQWS TYSMEDGHK YMEPSATFGHR GHLMEDFSAC HHFAASTR ALPMESS ????????K = Da ?????R = Da Slide 59: Mass of peptides compared to theoretical masses of all peptides known, using a computer program. Voice: The mass of each unknown peptide is put into a computer and compared to our list of known computer peptides. ???????? = Da

Mass of peptides matched to theoretical masses known peptides, using a computer program.
Computer Peptides WEGETMILK ADEMTYEK PLMEHGAK LMEHHH ASTEER DMGEYIILES EGEDMPAFY CYHGMEI EFPKLYSEK YSEPYSSIIR IESPLMIA AEFLYSR DLMILIYR METHIPEEK KISSMER PEPTIDEK MANYCQWS TYSMEDGHK YMEPSATFGHR GHLMEDFSAC HHFAASTR ALPMESS ????????K = Da ?????R = Da Slide 60: Mass of peptides matched to theoretical masses known peptides, using a computer program. Voice: During this comparison, we find that each of our unknown peptides match a known computer peptide. ???????? = Da

The unknown peptides have been identified
????????K = Da WEGETMILK ?????R = Da ASTEER Slide 61: The unknown peptides have been identified. Voice: These are the sequences of our mystery peptides. ???????? = Da MANYCQWS

Protein X has been identified
WEGETMILK AFTEER MANYCQWS Slide 62: Protein X has been identified Voice: If we put these peptides together we now know the sequence of protein X. Our protein is

Summary – tools to study proteins?
Proteins are digested into peptides Peptides are analyzed with a mass spectrometer Match observed peptide masses to theoretical masses of all peptides in database Assemble those peptide matches into a protein identification Slide 63: Summary – tools to study proteins? Voice:

Concluding points about Proteomics
Proteomics is the analysis of all proteins Interdisciplinary research Essential to both basic and clinical research Protein are the workhorses of the cell Discovery research – drugs and diseases Proteomics tools allow identification of proteins Slide 64: Concluding points about Proteomics Voice:

Questions Slide 65: Questions

Introduction to Proteomics

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