Proteome Assignment Test PDF

Proteome Variable - in different cell and tissue types in the same organism - in different growth and developmental stages of organism Dynamic - Disease state - Drug challenge - Growth condition - Stress  More about Proteomics Proteomics: is the study of total protein comple...

Proteome Variable - in different cell and tissue types in the same organism - in different growth and developmental stages of organism Dynamic - Disease state - Drug challenge - Growth condition - Stress  More about Proteomics Proteomics: is the study of total protein complements, proteomes e.g. from a given tissue or cell type. Definitions: - Classical: restricted to large scale analysis of gene products involving only proteins. - Inclusive: aims to identify all the proteins in a cell or organism including any posttranslationally modified forms, as well as their cellular localization. Function and interactions. Need for Proteomics  Why Proteomics? ⚫Protein function ⚫Protein post-translational modification ⚫Protein localization and compartmentalization ⚫Protein protein interactions ⚫Protein expression studies  Need for Proteomics ⚫ We have mainly four types of biological molecules: lipids, proteins, DNA (generic materials) and carbohydrate) ⚫ We take those proteins with long chains of amino acids all linked together through amino amide bond ⚫ We dissolve the peptides and run into mass spectrometer ⚫ We can get two different types of information while we run the peptides into mass spec ⚫ In MS1, we get individual information of peptides with individual signal of each peptide. It is known as MS1 spectra ⚫ In MS2, we hit some energy to the peptide molecule present in MS1 and get the fingerprints of individual peptide ⚫ We then go for the database search to know the name of the original proteins Need for Proteomics  Goal for Proteomics ⚫ Protein expression -Quantitative study of protein expression between samples that differ by some variable ⚫ Structural proteomics - Goal is to map out the 3-D structure of proteins and protein complexes ⚫ Functional proteomics -To study protein protein interaction, 3D structures, cellular localization to understand the physiological function of the whole set of proteome Scope of Proteomics?  Scope of Proteomics ⚫ Humans have 20,000 to 25,000 basic protein types. ⚫ Proteins can be studied in various contexts, including sequence, structure, interactions, expression, localization and modification. ⚫ Proteomics is divided into several major but overlapping branches, that embarce these different contexts: - Sequence or structural proteomics - Expression proteomics - Interaction proteomics - Functional proteomics Scope of Proteomics  Why Proteomics is important? ❑ Identification of proteins in normal and disease conditions - Investigating epidemiology and taxonomy of pathogens - Analysis of drug resistance ❑ Identification of pathogenic mechanisms - Reveals gene regulation events involved in disease progression ❑ Promise in novel drug discovery via analysis of clinically relevant molecular events ❑ Contributes to understanding of gene function Current Challenges of Proteomics?  Challenges Possible Solutions 1. Protein Solubility Develop a new top-down MS Conventional surfactants (e.g. SDS) compatible surfactant not compatible with MS 2. Proteome Complexity Develop novel multi- Intact protein chromatography under dimensional chromatography developed for intact protein separation 3. Proteome dynamic range Develop novel nanomaterials Difficulty in defecting low abundant for enriching low abundant proteins proteins 4. Protein MS data interpretation Develop user-friendly and Software for proteomics versatile software interface underdeveloped  L-2: 1. Current challenges of proteomics and possible solutions 2. Strategies for Protein Separation 3. 1D Gel Electrophoresis for proteins Revision Challenges Possible Solutions 1. Protein Solubility Develop a new top-down MS Conventional surfactants (e.g. SDS) compatible surfactant not compatible with MS 2. Proteome Complexity Develop novel multi- Intact protein chromatography under dimensional chromatography developed for intact protein separation 3. Proteome dynamic range Develop novel nanomaterials Difficulty in defecting low abundant for enriching low abundant proteins proteins 4. Protein MS data interpretation Develop user-friendly and Software for proteomics versatile software interface underdeveloped  Further Challenges ❑ Correlated biomarkers often do not improve disease prediction ❑ High Abundance of proteins in clinical samples ❑ False positive associations with proteomics data analysis Strategies for Protein Separation Proteomics Methodology 1D Gel Electrophoresis Basic Molecular Technique Spectroscopy Gel Electrophoresis Chromatography Blotting  Gel Electrophoresis Electrophoresis: Movement of any charged particle under an electric field Electric field: due to the application of voltage across the electrodes Opposite charge particles moves towards the electrodes Gel?? A semi-solid matrix or path where charged particles can move. It is a track where we can understand which is moving fast or slow.  1-Dimensional GE SDS-PAGE Native-PAGE Proteins are separated Proteins Separate by original in denatured forms native forms Proteins are treated No SDS treatment with SDS SDS: Sodium Dodecyl Sulfate 1. Anionic Detergent 2. Denaturant 3. Makes the denatured polypeptides/proteins net negatively 4. Charged by adding -2 charge  + SDS (net negatively charged) All the molecules will move towards the anode during electrophoresis SDS : Denatured the proteins and makes the negative charge to proteins  1-Dimensional Gel Electrophoresis A. SDS-PAGE: A.1. Separation of proteins on The basis of their molecular weight/size A.2. : Proteins are treated with SDS, beta- mercaptoethanol (beta ME) (breaks disulphide linkage) A.3. Heat (breaks the weaker interaction) BPB: tracking dye Glycerd: stacking agent CBB: Coomassie brilliant Blue  Polyacrylamide gel electrophoresis (SDS-PAGE) In separating the proteins by mass, the gel treated with sodium dodecyl sulfate (SDS) along with other reagents (SDS-PAGE in 1-D). This denatures the proteins (that is, it unfolds them into long, straight molecules) and binds a number of SDS molecules roughly proportional to the protein's length. Because a protein’s length (when unfolded) is roughly proportional to its mass, Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other.  Principle of SDS-PAGE In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium dedecyl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length.  Native PAGE 1. No denaturant added 2. Proteins separate in their native form (active form) 3. Useful in determining the mol wt. of active proteins and the no of subunits consisting the proteins By comparing the Native Page Pattern with the SDS-PAGE of a protein  What is electrophoresis? Migration of macromolecules (such as nucleic acids and proteins) in an electric field?  Polyacrylamide Gel Electrophoresis (PAGE) In PAGE, the separation of proteins depends on their 1. Charge density (charge-mass ratio) 2. Size (or Molecular Weight) and shape 1. Proteins carry charge positive due to amino group and negative due to –COOH group 2. Net Charge of a protein depends on pH (it can be negative, positive or neutral)  Charge density (Charge-to-mass ratio) 1. In PAGE, pH of the buffer is set such that all proteins at that pH will carry a negative net charge 2. Being negative charge, they will migrate to the anode (positive electrode) Higher charge density moves faster NATIVE PAGE Example Sub units: 4 Difference between Native and SDS-PAGE  L-3: 1. Revision of SDS PAGE 2. Native Polyacrylamide Gel Electrophoresis 3. Application of SDS PAGE and Native GE  Gel Electrophoresis: For Proteins 1-Dimensional GE 2-Dimensional GE Separation of proteins on Separation of proteins on the basis of their molecular the basis of their Isoelectric size /weight only point and molecular size /weight 1-Dimensional GE SDS-PAGE Native-PAGE NATIVE-PAGE  Types of Gels Agarose Polyacrylamide Matrix Matrix used as used as agarose polyacrylamide Agarose Gel Electrophoresis (AGE) Polyacrylamide Gel Electrophoresis Application: (PAGE) Separation of nucleic acids Application: molecules Separation of proteins + nucleic acid Sequencing  Agarose?? It is a polysaccharide, a constituent of agar composition Agarose Agaropectin Linear polymer of Agarobiose (disaccharide)  Polyacrylamide Polymer of acrylamide Highly toxic, carcinogenic Can be cross-linked by methylenebisarylamide PAGE: Used for Proteins and small Nucleic acids separation  Why Polyacrylamide?? Large pore size: Small pore size: Suitable for electrophoresis Suitable for electrophoresis of larger molecules like DNA of small molecules like proteins  Separation in PAGE Dependent factors: 1. Charge density (charge-to-mass ratio) 2. Size (or Molecular weight) and shape  Charge density (charge-to-mass ratio) Proteins are composed of amino acids Net Charge of a protein depends on pH (negative, positive or neutral) pH : In PAGE, the buffer is used to set the medium in such a way that all proteins at that pH will carry a negative net charge  Protein movement (charge-to-mass ratio) pH : In PAGE, the buffer is used to set the medium in such a way that all proteins at that pH will carry a negative net charge -ve Being negatively charged, they will migrate to anode (positive electrode + ve  PAGE: Charge density (charge-to-mass ratio) Protein 1-: Higher charge density Protein 2-: Lower charge density The greater the charge density, Rapid the rate of migration of proteins  PAGE: SIZE and SHAPE Size (Molecular weight): Depends on the number of amino acid units in a protein Shape Globular Elongated Depends on geometry how the protein exists  PAGE: SIZE -Ve The pore size of the GEL matrix Also affects the movements of proteins Large Larger the protein, Slower the migration Small Larger molecular size proteins are +Ve entangled with GEL pores  PAGE: Shape Globular proteins migrate faster than -Ve elongated proteins Elongated protein of comparable molecular weight Globular protein +Ve PAGE: NATIVE PAGE Protein migration dependent: 1. Charge density (charge-to-mass ratio) 2. Size and Shape Proteins remain in their NATIVE form NATIVE PAGE Advantages and disadvantages of NATIVE PAGE This method is preferred when our requirement is detection of particular proteins on the basis of biological activity, i.e. Separation and detection of Enzymes Limitation: It is not suitable for determination of molecular weights of proteins  L-4: 1. 2D Gel Electrophoresis 2D Gel Electrophoresis  Gel Electrophoresis: For Proteins 1-Dimensional GE 2-Dimensional GE Separation of proteins on Separation of proteins on the basis of their molecular the basis of their Isoelectric size /weight only point and molecular size /weight 1-Dimensional GE SDS-PAGE Native-PAGE  2D Gel Electrophoresis SDS PAGE: Separated based on Molecular weight of proteins Why 2D? Isoelectric point (PI) Separation Molecular Weight (MW) Requirement of 2D PAGE: Situation: Two proteins come with the same/similar molecular wt in GE 1D GE 2D GE Need to consider two parameters for separations X  What is Isoelectric Point (pI)? - ve pH=14 pKa1 pKa2 GE pH=7 Low pH High pH pH=1 (protonated) (Deprotonated) + ve (Anode) An isoelectric point (pI) is a point where the new charge of a protein = 0 pK1 and pK2 of amino acids  Principle of 2D gel electrophoresis First Dimension: Protein separation according to isoelectric point Container - + Proteins pH=11 pH=3 Strip Proteins movement towards isoelectric point  Principle of 2D gel electrophoresis Second Dimension: Protein separation according to Molecular Weight Principle of 2D gel electrophoresis END  L-5: 1. Application of 2D Gel Electrophoresis  Applications of 2D GE ❑ Protein profiling-a global view of proteins of interest Profile your sample. Protein profiling-a global view of proteins of interest ❑ Differential display-biomarker discovery If you consistently can do protein profile then you can move on for differential display where you have different treatment of your sample. You can find the difference in proteins of your samples and find the potential biomarkers related to whatever mechanism you are done ❑ Protein posttranslational modification-protein function During post translational modification, you are changing the pI of protein and resulted that is that you can find the changes in a different spots location on the gel Principles of Multidimensional Liquid Chromatography Liquid Chromatography-Mass Spectrometry LC-MS  Invention of Chromatography It was Mikhail Tswett, a Russian botanist, in 1903 who first invented and named chromatography Tswett used a glass column filled with finely divided (calcium carbonate) to separate plant pigments. He observed the separation of colored zones or bands along the column. The development of chromatography was slow and scientists waited to early fifties for the first chromatographic instrument to appear in the market (a gas chromatograph). liquid chromatographic equipment with acceptable performance was only introduced about two decades after gas chromatography  Chromatography ❑ The separation of a mixture by distribution of its components between a mobile and stationary phase over time. ❑ mobile phase = solvent (also called eluent) penetrates or passes through a solid or immiscible stationary phase ❑ Stationary phase = column packing material ❑ In a chromatographic separation of any type, different components of a sample are transported in a mobile phase (a gas, a liquid, or a supercritical fluid)  Stationary Phase and Mobile Phase Since the stationary phase is the fixed one then those solutes which have:- stronger interactions with the stationary phase will tend to move slower (have higher retention times” The time a solute spends in a column”) than others that have lower or no interactions with the stationary phase will tend to move faster. chromatographic separations are a consequence of the differential migration of solutes maximum interactions between a solute and a stationary phase take place when both have similar characteristics, for example in terms of polarity. Details of Liquid Chromatoghraphy  Partition Chromatography/ Liquid-Liquid chromatography How was Partition Chromatography developed? Hydrophilic compound remains in aqueous phase Hydrophobic compound remains in aqueous phase Solvent Extraction method Distribution of compounds between two phases depends on the surface area between two liquids Surface area Separation  Partition Chromatography/ Liquid-Liquid chromatography Chemically modified silica is kept to hold the liquid stationary phase Chromatography Column Sample When sample is loaded, the sample molecules dissolves in liquid phase Partition Chromatography/ Liquid-Liquid chromatography Mobile phase is a solvent or a mixture of solvent immiscible with stationary phase Sample molecules at stationary phase (liquid) interact with Mobile phase (liquid) Interact more with aq. Phase Interact more with mobile phase Sample molecules will have different rate of migration in column Partition Chromatography/ Liquid-Liquid chromatography Sample molecules are collected as fraction during chromatography Chromatography fractions Specific volume of mobile phase solvent (Eluate) collected in tubes after chromatography as termed as fraction Liquid Chromatography can be used for separation of various proteins and enzymes L6: HPLC  Liquid Chromatography (LC) According to the nature of the mobile phase, chromatographic techniques can be :- * classified into three classes: a. Liquid chromatography (LC) b. Gas chromatography (GC) c. Supercritical fluid chromatography (SFC) Liquid Chromatography (LC) Column Higher performance Thin layer (gravity flow) (pressure flow) (adsorption) 77  Higher performance/pressure Liquid Chromatography Higher Pressure pump Mobile phase Modified column chromatography Sample Stationary phase (silica, alumina, cellulose) Liquid-liquid chromatography: Mobile phase is passed through the column due to gravity HPLC: 1. High pressure of about 40 kilopascal is generated in column 2. Columns are filled with stationary phase adsorbent materials with small particle size 3. Small particle size offers large surface area 4. Finally it gives high resolution  HPLC: Configuration of column 5-25 cm Stainless steel ❑ Stainless steel, Can tolerate high pressure ❑ 4.5 mm of internal diameter ❑ Flow rate of mobile phase 1-3 ml/min’ ❑ Stationary phase: Stationary phase adsorbent materials- very small size chemically modified silica divinyl benzene etc.  HPLC: Mobile Phase High Pressure Pump Sample injector Solvent: Polar or non-polar Solvent selection: Depends on type of samples Mobile phase is connected with a pump which can flow the solvent to column  HPLC: Detector UV detector Detector IR deetctor Refractive index detector Mass Spec Electrochemical detector  HPLC: Interpretation data Unknown: Glucose present, Sucrose absent HPLC: Unknown concentration determination Explained on board  L7: 1. HPLC pumps 2. Liquid chromatography 3. Multidimensional Chromatography LC-MS Workflow  HPLC PUMPS Autosampler Degasser for removing any Mobile phase A: Usually Aqueous phase gas from the line. Mobile Phase B: Usually Organic phase We have a binary pump system and the purpose of binary pumps for 1. To deliver mobile –phase at a steady flow 2. Precise amounts of solvents should be mixed Before entering to columns  Process of separation in LC-MS A small volume of the sample is first introduced at the top of the chromatographic column. Elution involves:-  Process of separation in LC Passing a mobile phase inside the column whereby solutes are carried down the stream but on a differential scale due to interactions with the stationary phase. As the mobile phase continues to flow, solutes continue to move downward the column. Distances between solute bands become greater with time and as solutes start to leave The column they are sequentially detected. The following schematics represent the process at various times:  Separation in column chromatography The dark colors at the center of the solute zones in the above figure represent higher concentrations than are concentrations at the sides. This can be represented schematically as:  Parameters The plot of the detector signal versus the retention time of solutes in a chromatographic column is referred to as a chromatogram The areas under the peaks in a chromatogram are usually related to solute concentration “quantitative analysis” The retention time of a solute is a characteristic property of the solute which reflects its degree of interaction with both stationary and mobile phases. Retention times serve “qualitative analysis” parameters to identify solutes. Basic Liquid Chromatography Mobile Phase supply system Programmable pump Sample Valve Column Detector Data Interpretation tool Basic Liquid Chromatography  The Mobile Phase Supply System The mobile phase supply system consists of number of reservoirs (200 ml to 1,000 ml in capacity)  The Gradient Programmer and the LC Pump -the solvent mixing occurs at high pressure and then passed to the pump -is the simplest but most expensive. -each solvent requires its own pump.  The Sample Valve in LC-MS -liquid samples are usually injected onto the column by a syringe via a injector. -Sample are placed on an LC column directly with either an internal or external loop sample valve the valve being connected directly to the column. Combine LC with Mass Spectrometer: LC-MS * combination of LC and MS offers the possibility to take advantage of both LC as a powerful and versatile separation technique and MS as a powerful and sensitive detection and identification technique. a mass spectrometer is more sensitive and far more specific than all other LC detectors. -It can analyze compounds that lack a suitable chromophore. -It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography. Mass spectral data complements data from other LC detectors Two-dimensional abundance data and three-dimensional mass spectral data from a mass spectrometer  Multidimensional Chromatography 1. The first recorded use of multidimensional chromatography is believed to have been conducted by R. Consden, who successfully separated out 22 hydrochlorides from amino acids using paper chromatography. 2. Multidimensional chromatography is a chromatographic technique that is capable of delivering heightened separation performance for complex and difficult substances. 3. There are a whole host of highly complex mixtures which are desirable to be separated for qualitative and quantitative analysis, but simply can’t be due to their complex nature Multidimensional chromatography can be used for a wide variety of applications, including: ❑ Identification of proteins and peptides ❑ Identification of DNA fragments ❑ Determination of biomarkers in petroleum and oil ❑ Drug isolation in urine ❑ Drug isolation in plasma ❑ Refinement of surfactants L-8: Proteins Identifications ❑ Western blotting Technique ❑ Advantages and disadvantages  Strategies for Protein Identification: Blotting is a technique for the detection of macromolecules 1. Southern blotting: DNA detection 2. Northern blotting: RNA 3. Western blotting: Protein identification Western blotting : A. Northern blotting and southern blotting are almost similar techniques where we need similar types of setup B. Western blotting is different than above mentioned blotting techniques Advantages of western blotting : A. Western blotting can identify nanograms of proteins molecules in cell fraction or mixture B. A widely used technique for the detection of various reactions within the cells and identify various proteins within cells  Western blotting : A. Any blotting process relies on three steps: Step-1 :Extraction of the molecules. Step-2 : Electrophoresis Step-3: probing Here molecules mean proteins. We extract the proteins contained in the cells. We first treat the cells with detergent so that the cells membranes should be broken and the cytosolic compounds should come out. We do for centrifuge and the supernatant should be the proteins of the cells. Separation of proteins SDS PAGE Separation By electrophoresis Basis: Mass Proteins mixture and shape of proteins Smaller proteins will migrate faster than larger proteins Why membrane? We want only the Transfer of the target proteins should gel contents onto bind to probe, but not a nitrocellulose other regions of gel. membrane/PVDF  Western blotting : How to transfer the proteins from the gel to the membrane? - - - - - - Gel Direct contact Membrane with the gel + + + + + + Protein molecules will migrate from gel to membrane Blotting (transfer): Transfer of protein molecules from gel to membrane  Western blotting: After transfer from Gel to membrane Region of proteins Third stage: Probing and Hybridization Membrane We need some detectors that can bind and detect particular proteins Therefore we need antibodies so that they can bind our target proteins in nitrocellulose membranes Blocking agent addition. Antibodies can bind unspecific areas. To reduce the background noise, we use a blocking agent.  Western blotting: 1. Primary Ab is added first to directly bind to target proteins. But, it can not show any results as it does not show any fluorescence Or visible light to identify the proteins Wash out the medium to remove unwanted antibodies ❑ Next: Add a secondary antibody. It acts as a reporter. ❑ Secondary Ab is targeted to capture the 1st Ab. ❑ Secondary antibody has enzyme ❑ If we add the substrate which converts to products and provides the fluorescence light Enzyme used here is called “horseradish peroxidase” (HRP). It produces light and we can see the particular proteins  Steps in Western blotting ❑ Protein Extraction: Proteins are extracted from cells after rupturing of the tissue or cells ❑ Protein Separation: Extracted proteins are separated through SDS-PAGE ❑ Protein Transfer: After separation, proteins are transferred to the nitrocellulose membrane ❑ Blocking: To reduce background noise and non-specific binding of antibodies, We use bovine serum albumin (BSA) or dry milk to saturate the unoccupied binding area of the membranes ❑ Antibody incubation: Primary antibodies specific to the target protein are added. ❑ Washing: Membranes are washed to remove the unbound primary antibodies ❑ Secondary antibody incubation: This is conjugated with an enzyme and binds to the primary antibody ❑ Detection: The target proteins are visualized after adding a substrate which results fluorescent signal  Advantage of Western blotting ❑ Protein specificity: You can detect the specific proteins ❑ Quantitative analysis: If you compare the signal intensities With a reference protein (control), you can estimate the relative the abundance of the proteins ❑ Low sample requirements: You can detect very quantity of proteins through this technique Disadvantage of Western blotting ▪ Time-consuming: ▪ Sensitivity: It is not sensitive like enzyme-linked immunosorbent assay (ELISA) or mass spectrometry. ❑ Not digital technique. It involves chemiluminescent or fluorescent detection for which you need a special type of equipment L-9: Proteins Identifications ❑ Protein sequencing ❑ Sanger’s method ❑ Edman Method  How to determine Amino acid sequence of polypeptide chain? How we know? Phenylalanine(phe) bound to Leucine (Leu) bound to Serine (Ser) bound to Cystine (cys) ❑ Amino acid sequence is determined by protein sequencing ❑ Protein sequencing refers to methods for determining the amino acid sequence of proteins (or peptides) and analysis of the sequence  Concept of N-terminal and C-terminal Tripeptide chain 1 3 2 COOH group present is called the “C-terminal” “NH2” group is present is called the “N-terminal” Peptide chain of 7 amino acids  Protein sequencing N-terminal analysis C-terminal analysis Amino-terminal end of polypeptide Carboxy terminal end of polypeptide Chain determined chain determined ❑ Sanger’s Method ❑ Carboxypeptidase Method ❑ Edman’s Method Sanger’s Method (N-terminal analysis) (We will react with polypeptide to whom we will make the sequence) We use either acid or enzyme for hydrolysis of peptide bond Separated and identified 2,4-Dinitrofluro- Polypeptide Dinitro phenyl (we can separate by different benzene (DNFB) Polypeptide (DNP) chromatographic or relevant (yellow derivative) techniques)  Edman’s Method (N-terminal analysis) The Edman degradation technique, developed by Pehr Edman in the 1950s, is a widely used method for sequencing the amino acids in a peptide or protein. Also known as Edman’s Degradation Uses phenylisothiocynate (Edman’s reagent) Sequentially removes one residue at a time from the amino end of a peptide (N terminal) (From this, we can understand which amino acid was bound to a particular amino acid) Edman’s Method (N-terminal analysis) Stable  Step by step Edman’s Method 1. Reaction been Edman reagent And peptide happen in mild alkaline medium 2. Product is phenyl thocarbamoyl derivative Label and cleave Phenyl isothiocynate (Edman’s reagent) Extracted by solvent Advantages and Disadvantages of Edman’s Method Advantages: ✓ We can go for sequential amino acid determination from the N-terminus polypeptide chain ✓ It is high precision technique ✓ Edman degradation may work for small amino acids ✓ It is a well-established method Disadvantages: We can not use this technique for non-N terminus amino acid protein sequencing It is time consuming Mass Spectrometry Principles & Instrumentation  What is mass spectrometry ? WEIGHING MOLECULES Scientists use mass spectrometry to weigh molecules. Molecules are extremely small and cannot be weighed in the traditional sense on a scale or by implementing gravitational force. Gravitation 3 keV (electronvolt) 1 km flight path 0.8 mm neutral molecular beam ►The interaction between neutral species and gravity is relatively weak; a 3 keV neutral molecular beam of mass range 100 to 200 Daltons is gravity broadened by only 0.8 mm over a 1 km flight path. ►In order to achieve mass separation of molecular or atomic species on a practical scale it is necessary to ionize the species under investigation so one can take advantage of the basic principles of ion physics for ion separation and analysis. 5  J. J. Thomson – Parable Spectrograph Ion Source Magnetic and Electric Field Photographic Plate First mass spectrum of 20Ne and 22Ne 6  Goals of Mass Spectrometry Determine molecular weights (MW) from small molecules to large polymers (>100000Da) Provide quantitative information Down to sub part-per-trillion (ppt) concentrations of analytes in complex mixtures Elucidate compositional and structural details of investigated species Mismatch of one aminoacid in a sequence of large proteins could be determined  Areas for MS applications Proteomics Glycobiology Genetics and medicine Molecular and cell biology Environmental sciences Agriculture Polymer chemistry Antiterrorism Components of mass spectrometry  High Vacuum In a mass spectrometer, the mean free path of ions (the distance the ion must travel without collision with gas molecules before detection) ranges from centimeters to kilometers, therefore the maximum pressure required for a specific mass spectrometer is usually between 10-3 to 10-9 Torr. Pressure is controlled utilizing efficient pumping systems involving mechanical pumps in conjunction with turbo-molecular pumps. High Vacuum Mass Spectrum... Mass Spectrum A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted to ions; i.e., molecules that have been electrically charged. mass spectrum Mass spectrometry is used to determine: 1) Molecular weight 2) Molecular formula 3) Elemental composition 4) Structure 5) Identify unknown compounds, 6) Quantify known compounds etc. L10 and 11: Mass Spectrometry Principles & Instrumentation  Mass Analysis is like sorting and counting 1. Pocket change (mixture of coins) 1. Mixture of molecules 2. 50 ps, 1 Rs, 2 Rs, 5 Rs, & 10 Rs 2. Molecules of different weight, size 3. Sorting change by value or size 3. Separation by mass & charge 4. Concept of visual interpretation 4. spectrum  Methods for generating ions for mass spec 1. Electron impact ionization 2. Chemical ionization 3. Fast atom bombardment 4. Electrospray ionization Electron impact ionization =0.14 nm  Probability of electrons breaking chemical bond is maximum. If the kinetic energy of electron is less than 70 eV, the probability of breaking the chemical bond will be less  Electrospray ionization 1. Sample molecules are sprayed in the electric field to produce ions 2. Popular for analysis of biomolecules: protein and DNA L-12: Various parameters associated with mass spectrometer including the peaks, and interpretation, mass accuracy, quadrupole mass spectrometer Mass resolution Mass resolution R = m/ ∆m ∆m=FWHM ∆m R = m/ ∆m Full Width at Half Maximum Mass resolution and peak separation Mass resolution: peak shape  Mass resolution of small ions C2H6+ (m/z = 30.046950)C2H6, CH2O, and NO all have a unit mass/charge (m/z rounded to the nearest whole number) of 30. A low resolution mass + CH2O (m/z = 30.010565) spectrometer (e.g., quadrupole) can distinguish ions with the NO+ (m/z = 29.997989) same unit m/z. High resolution mass spectrometers (e.g.,TOF), are capable of + + + resolving MS peaks corresponding to C2H6 , CH2O , and NO and identify the ions based on their "exact mass". NO+ CH2O+ Mass resolution ∆m R = m/ ∆m ∆m=FWHM + C2H6 Full Width at Half Maximum 22 29.997989 30.010565 30.046950 L-12: Mass spectrometer analysis for protein identification  Work Flow & Instrumentation Protein Separation / Mass Spectrometry / Interpretation Extraction Derivitization Ionization Cells Electrophoresis TOF Protein Identification Tissues Chromatography Quadrupole Differential expression profile Organs ICAT Ion-Trap Others Sequence Biological Fluids FTMS comparison Hybrid Differential MS & MS/MS modification analysis ESI MALDI  Mass Table Element / Nominal Average Isotope Exact mass % natural Molecule mass mass abundance Hydrogen – H 1 1.00794 1H 1.00783 99.984% 2H 2.01410 0.016% Carbon – C 12 12.01115 12C 12.00000 98.9% 13C 13.00336 1.1% Nitrogen – N 14 14.0067 14N 14.00307 99.62% 15N 15.000109 0.38% Oxygen - O 16 15.9994 16O 15.99491 99.76% 18O 17.99915 0.20% Water (H2O) 18 18.01528 1H16O 18.01057 2 Isotopes Analysis by Mass Spectrometry ❑ Distinguishes different isotopes of a given element ❑ More peaks may be visible with m/z larger than the molecular ion peak due to isotope distributions ❑ Methane (CH4) approximately 1 in every 100 contains than carbon-13 carbon-12 ❑ It means 1 in every 100 of the molecules will have a mass of 17 (13 + 4) rather than 16 (12 + 4). ❑ Mass spectrum will have the line corresponding to the molecular ion [12CH4]+ as well as [13CH4]+. ❑ Molecular peak (M+) of the [12CH4]+ is going to be at m/z 16 and the [13CH4]+ (isotope) peak (M+1) will be at 17 but the intensity of M+1 peak will be 1.1% of the [12CH4]+ ion Mass spectrum thus can give a clue on the number of carbon atoms present in a molecule ❑ Ethane (C2H6) either of its 2 carbon atoms has an approximately 1 in 100 chance of being 13C ❑ Therefore a 2 in 100 chance of the molecule as a whole containing one 13C atom rather than a 12C ❑ M+ ion of ethane will be at m/z 30 and the M+1 peak will be at m/z 31 but having a height of 2.2% of the M+ peak ❑ The chances of both the carbon atom of ethane having 13C atom is 0.0001% (0.011 x 0.011) and is negligible. ❑ Propane (C3H8) either of its 3 carbon atoms has an approximately 3 in 100 chance of being 13C ❑ Therefore a 3 in 100 chance of the molecule as a whole containing one 13C atom rather than a 12C ❑ M+ ion of propane will be at m/z 44 and the M+1 peak will be at m/z 45 but having a height of 3.3 % of the M+ peak ❑ The chances of all three carbon atoms of propane having 13C atom is 0.00001% (0.011 x 0.011 x 0.011) and is negligible. Mass spectrum isotope distribution in bromoethane (CH3Br) Natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu Natural chlorine (Cl) consists of 75% of 35Cl and 25% of 37Cl. The mass spectrum of Bromomethane (CH3Br) The molecular ion peaks of CH3Br each contain one Bromine atom - but the bromine can be either of the two bromine isotopes, 79Br and 81Br as mentioned above. Therefore, the mass spectrum shows molecular ions at m/z 94 and m/z 96 at almost equal heights or intensity because both 79Br and 81Br are in equal proportions in nature  Nominal mass What is Nominal Mass? ❑ Mass of a molecular ion or molecule is calculated using the isotope mass of the most abundant constituent element isotope of each element rounded to the nearest integer value and multiplied by the number of atoms of each element. ❑ Nominal mass is calculated using the integer mass of the most abundant isotope of each element.  Exact mass, average mass, and accurate mass What is Exact mass? Exact mass is the mass of the most abundant isotope species or individual isotope species of an element or molecule. The exact mass is the calculated mass of an ion or molecule with a specified isotopic composition. Exact mass of an ion or molecule calculated using the mass of the most abundant isotope of each element is called monoisotopic mass Average mass is the mass of an ion or molecule weighted for its isotopic composition. In other words average mass is the weighted mean of all the isotopic species in a molecule. It is calculated using all isotopes of each element and their natural abundance Accurate mass is the experimentally determined mass of an ion (of known charge). Please note that accurate mass and exact mass are not synonymous. Accurate mass refers to a measured mass, and exact mass refers to a calculated mass  Mass Resolution & Mass Accuracy High-Resolution mass spectrometer (HRMS) can separate the molecules by exact mass ❑ Compounds having similar nominal masses can be separated and identified using an HRMS. For example both propane (CH3CH2CH3) and acetaldehyde (CH3CHO) have nominal mass as 44. However, by using an HRMS both can be distinguished because their exact masses (or monoisotopic mass) are 44.0626 and 44.0262 for CH3CH2CH3 and CH3CHO respectively. The better the resolving power of an HRMS, the more the separation of the ions. Resolving power is the measure of the ability Resolving power of a mass spectrometer to provide a specified value of mass resolution. Mass resolution in a mass spectrum is the observed m/z value divided by the smallest difference Δ(m/z) for two ions that can be separated = (m/z)/Δ(m/z). This is performed using peak width measured at a specified percentage of peak height, generally at 50% peak height, called full width at half maximum (FWHM). See Figure to appreciate how resolving power is calculated, in which the resolving power (RP) is 5000.  Mass Error ❑ Even with the best HRMS, the accurate mass (measured mass) of a molecular ion will be slightly different than the exact mass (or more precisely, monoisotopic mass). This difference observed in the measured mass is called “Mass Error” ❑ Mass error = (exact mass) – (accurate mass) Mass error is expressed in parts per million (PPM) = ❑ The lower the PPM, the better the mass accuracy of a mass spectrometer.  Mass resolution of small ions C2H6+ (m/z = 30.046950)C2H6, CH2O, and NO all have a unit mass/charge (m/z rounded to the nearest whole number) of 30. A low resolution mass + CH2O (m/z = 30.010565) spectrometer (e.g., quadrupole) can distinguish ions with the NO+ (m/z = 29.997989) same unit m/z. High resolution mass spectrometers (e.g.,TOF), are capable of + + + resolving MS peaks corresponding to C2H6 , CH2O , and NO and identify the ions based on their "exact mass". NO+ CH2O+ Mass resolution ∆m R = m/ ∆m ∆m=FWHM + C2H6 Full Width at Half Maximum 22 29.997989 30.010565 30.046950  Accuracy of Mass Measurment Radical ion of ethylamine (C2H5NH2. +) Mass of the ion 2 · 12C + 7 · 1H + 14N Unit or Nominal mass of the ion 2 · 12 + 7 · 1 + 14 = 45 Exact (nominal) mass of the ion 2 · 12.000000 + 7 · 1.007825 + 14.003074 = 45.057849 45.057889 Mass accuracy (ppm) 45.057849 ((Mm-Mn)/Mn) x 1,000,000 Mm - measured mass (45.057889) Mn - nominal mass (45.057849) Mass resolution and peak separation Mass resolution: peak shape Mass Spectrometer Mass Spectrometer 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 L-13: Protein Array  What is a protein array? Proteomics: ❑ A protein array is a high throughput method for the study of the interaction, function and activities of proteins ❑ It involves immobilizing multiple proteins on a solid surface, such as slide and membranes so that researchers can test the interaction with other biomolecules Why Important? ❑ This technique is commonly used in proteomics, the large-scale study of proteins, to explore functions, interactions, or disease-related changes.  Protein Array: Introduction Proteomics: ❑ The study of all the proteins that are expressed at a certain time inside cells, tissues, organs, organisms ❑ Protein profiling includes: How abundance of proteins, how interactions, activity and about structural modification take place in the protein these all things come under protein profiling under proteomics. Goals: To do this fast and accurately Proteomic methods 2D-Gel LC/MS Microarrays Electrophoresis  Protein Array Micro array technology was first developed from an earlier concept called “Ambient analyte immunoassay” by Roger Ekins in 1989 Later transformed into DNA microarray Due to some limitations in DNA microarray, this protein microarray was developed to overcome those limitations Concentrations of mRNAs within a cell are poorly correlated with actual abundances of the corresponding proteins  Protein Microarray (protein chip) A protein microarray is a high throughput method used to track interactions and activities of proteins, and to determine their function, and determining function on a large scale Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by laser scanner  Types of Protein Arrays 1. Analytical Protein Arrays Used for detecting the presence or concentration of proteins (e.g., antibodies, cytokines). Applied in disease biomarker discovery and diagnostics. Example: Antibody arrays. Analytical protein arrays are primarily used to detect and quantify proteins (such as antibodies, antigens, or cytokines) in complex biological samples, including serum or plasma. They focus on diagnostics and biomarker discovery by measuring changes in protein levels or their presence/absence. Workflow of Analytical Protein Arrays 1. Array Preparation: Antibodies are immobilized on a solid surface. 2. Sample Incubation: Biological samples (e.g., serum) are applied to the array. 3. Washing and Blocking: Removes non-specific interactions. 4. Detection: Labeled secondary antibodies or other detection molecules are used. 5. Signal Measurement: Signals (fluorescence, colorimetric, or chemiluminescence) are recorded. 6. Data Analysis: Signals are quantified to determine the concentration or presence of target proteins. This approach allows researchers to perform comprehensive, multiplex protein analysis, facilitating early diagnosis and personalized medicine.  Types of Protein Arrays Functional Protein Arrays Consists of purified proteins and are used to study enzymatic activities, interactions, or binding properties. Help identify protein-protein, protein-DNA, or protein-ligand interactions. Useful for drug screening and pathway analysis. Reverse-phase Protein Arrays (RPPAs) In this setup, samples (e.g., cell lysates) are spotted on the array, and antibodies are used to detect specific proteins. Commonly used in clinical research, especially in cancer studies, for profiling protein expression and post-translational modifications.  Workflow: Protein Microarray 1. Array Design and Selection Identify the purpose of the experiment (e.g., protein-protein interactions, immune profiling, biomarker discovery). Select the type of array: Antibody array Functional protein array Reverse-phase protein array (RPPA) 2. Protein or Antibody Immobilization Prepare the array surface (e.g., glass slide, nitrocellulose membrane, or gold-coated slide). Immobilize proteins (or antibodies) on the surface: Spot proteins/antibodies using robotic printers. Maintain proper spatial separation for downstream analysis. Ensure proteins are fixed in a way that retains their activity and functionality. 3. Blocking Non-Specific Binding Incubate the slide with a blocking buffer to prevent non-specific binding of detection molecules. Common blocking agents: BSA (Bovine Serum Albumin), skim milk, or casein. Reduces background noise and improves signal clarity. 4. Sample Preparation Prepare biological samples (e.g., serum, plasma, cell lysates). Label target proteins or molecules (e.g., with biotin, fluorescence, or enzymatic tags). If working with reverse-phase arrays, proteins or antibodies from the sample are spotted directly onto the surface. 5. Incubation Apply the labeled samples or target molecules onto the microarray. Incubate the array under optimized conditions to allow interaction between immobilized and target proteins (or antibodies). Wash off unbound materials to minimize background interference. 6. Detection and Signal Development Detect bound molecules using: Fluorescence scanners (for labeled molecules). Colorimetric detection (enzymes such as horseradish peroxidase). Chemiluminescence or radioactive labeling. 7. Data Acquisition Use a microarray scanner (such as fluorescence or CCD-based imaging) to detect and measure signals. The intensity of the signal correlates with the amount of binding or interaction between molecules. 8. Interpretation and Reporting Interpret the data to draw conclusions about the: Protein-protein interactions Biomarker discovery Disease mechanism or pathway analysis Report findings through detailed graphs, heatmaps, or pathway diagrams. Workflow Summary Diagram: 1.Array Preparation → 2. Protein Immobilization → 3. Blocking → 4. Sample Incubation → 5. Washing 2.Signal Detection → 7. Data Acquisition → 8. Data Analysis → 9. Validation → 10. Interpretation & Reporting  Workflow: Protein Microarray ❑ Proteins microarray is prepared by immobilizing proteins onto a microscope slide ❑ A variety of slide surfaces can be used such as glass light, nitrocellulose membrane bead, or micro-treated plate to which an array of capture proteins is bound. For example capture antibodies as shown in the diagram. Each spot represents one antibody that binds to a specific target protein Incubate protein sample as little as 10 microliter During sample incubation the capture antibody will bind to a specific protein Unspecific proteins should be washed off Incubate a cocktail of biotinylated detection Abs Immobilized antibodies Workflow: Protein Microarray Applications of Analytical Protein Arrays 1.Diagnostics: 1. Identification of disease-specific biomarkers (e.g., cancer markers, inflammatory cytokines). 2. Detect autoimmune disease-related antibodies. 2.Immunological Studies: 1. Profiling immune responses by measuring multiple cytokines. 3.Clinical Research: 1. Monitoring treatment efficacy by observing changes in biomarker levels. 4.Allergen Testing: 1. Detecting allergen-specific IgE antibodies in allergic individuals.  Advantages of Protein Microarrays High-Throughput Analysis: ❑ High-Throughput Analysis: Allows simultaneous analysis of thousands of proteins or interactions on a single array, saving time and resources. ❑ Small Sample Volumes: Requires minimal amounts of reagents and samples, making it ideal for experiments with limited sample availability. ❑ Comprehensive Profiling: Facilitates the profiling of entire proteomes, enabling the detection of low-abundance proteins and the exploration of multiple interactions (e.g., protein-protein, protein- DNA). ❑ Versatility: Disease biomarker discovery, Immune profiling, Drug screening, Functional studies (enzymatic activity or ligand-binding analysis)  Disadvantages of Protein Microarrays 1. Protein Stability Issues: Immobilized proteins may lose their natural conformation and activity, leading to false-negative results. 2. Non-Specific Binding: Despite blocking steps, non-specific interactions may occur, leading to background noise and false positives. 3. Limited Dynamic Range: The intensity of signals may saturate at high protein concentrations, leading to a limited range of accurate detection. Protein microarrays offer an excellent platform for high-throughput protein analysis, with applications in biomarker discovery, immune profiling, and drug screening. However, challenges such as protein stability, non-specific interactions, and data complexity need to be carefully addressed to ensure reliable results.  How Multiplexed Gel Electrophoresis Works? Gel Electrophoresis: All labeled protein samples are mixed and loaded onto the same 2D gel (IF + SDS-PAGE). As proteins migrate through the gel, each sample can be detected separately based on its dye fluorescence. Image Acquisition and Analysis: The gel is scanned at multiple wavelengths to detect signals from each dye. Image analysis software detects protein spots and quantifies differences in spot intensity between samples.  Difference Gel Electrophoresis (DIGE) ❑ Allows for simultaneous separation of up to 3 samples on one gel ❑ Monitor differences in proteomics profile between cells in different functional states ❑ Brings a new level of statistical confidence and reliability to 2D gel electrophoresis Step-1: Samples tagging with fluorescent dyes  Difference Gel Electrophoresis (DIGE) Step-2: Sample running Different samples co-migrate in 2D Gel Step-3: gel fluorescent imaging superimposing After run, different fluorescent images are superimposed on each other  Difference Gel Electrophoresis (DIGE) ❑ Fluorescent dyes designed in a way that minimally influences the migration behaviour of proteins and allows for the detection of the lowest quantifications Advantages: DIGE minimizes gel-to-gel variation and provides accurate relative quantification by directly comparing the intensity of fluorescent signals from different samples on the same gel. Applications: DIGE is commonly used for comparative proteomics to identify differences in protein expression between different conditions or experimental groups.  Applications of Multiplexed Gel Electrophoresis in Proteomics 1. Biomarker Discovery: Identifies proteins with altered expression between disease and healthy states. 2. Drug Mechanism Studies: Tracks changes in protein expression in response to drug treatments. 3. Comparative Proteomics: Studies differences across multiple biological conditions (e.g., cancer vs. normal tissue).  Advantages of Difference Gel Electrophoresis (DIGE) Reduced Experimental Variability: Since multiple samples run on the same gel, the method avoids issues with inter-gel variability. Direct Comparison Between Multiple Conditions: Allows simultaneous comparison of control vs. treated or healthy vs. diseased samples. Minimal Sample Requirement: Multiple conditions can be tested using smaller amounts of protein.  Limitations: Protein coverage limitations: Difficult to detect very low-abundance or hydrophobic proteins. Labor-intensive: Gel preparation, staining, and spot picking are time-consuming. Limited dynamic range: Compared to modern LC-MS-based methods. Possible solutions Combination with Mass Spectrometry: After spot picking, protein identification is done using MALDI- TOF or LC-MS/MS, enhancing the utility of multiplexed gels.