Protein Separation and Identification Techniques PDF
Document Details
Uploaded by LavishDenouement2766
Faculdade de Medicina Dentária
2024
Ana Peixoto Gomes
Tags
Summary
This document provides a lecture on protein separation and identification techniques. It covers topics such as proteome complexity, ELISA, multiplex immunoassays, and mass spectrometry-based proteomics. The lecture notes are from the 2024/2025 academic year and were given by Ana Peixoto Gomes
Full Transcript
Protein Separation and Identification Techniques Ana Peixoto Gomes 2024/2025 Protein Separation and Identification Techniques Proteome complexity Post-translational Genome...
Protein Separation and Identification Techniques Ana Peixoto Gomes 2024/2025 Protein Separation and Identification Techniques Proteome complexity Post-translational Genome Alternative promoters Transcriptome modifications (PTM) Proteome ~20-25 000 genes Alternative splicing mRNA editing ~ 100 000 transcripts > 1 000 000 proteins How does it start? Proteomics workflow - SAMPLE - EXTRACTION - SEPARATION - DETECTION - IDENTIFICATION - FUNTIONAL ANALYSIS, STRUCTURE,… HOW???? Low-throughput methods Chromatography-based methods Proteomics Gel-based methods Techniques Antibody-based methods High-throughput methods Mass spectrometry-based proteomics Enzyme-linked immunosorbent assay (ELISA) An immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples The term ELISA was first used by Engvall &Perlma in 1971. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. Nowadays is used for HIV diagnosis infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum HOW?? ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment. Each ELISA measures a specific antigen, and kits for a variety of antigens are widely available. 1. Antigen of interest is absorbed on to plastic surface (“sorbent”) 2. Antigen is recognised by specific antibody (“immuno‟). 3. This antibody is recognised by second antibody (2immuno‟) which has enzyme attached (“enzymelinked”). 4. Substrate reacts with enzyme to produce product, usually coloured. Type of ELISA Alhajj M, Farhana A. Enzyme Linked Immunosorbent Assay. [Updated 2021 Feb 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK555922/ https://www.youtube.com/watch?v=CWkrQrq0yxQ ELISA Qualitative/Quantitative Qualitative - determines antigen or antibody is present or absent Quantitative - determines the quantity of the antibody -Titer -The highest dilution of the specimen usually serum which gives a positive reaction in the test Multiplex immunoassay Bead-based immunoassay that allows for multiplex detection of up to 100 analytes simultaneously. Multiplex immunoassay Color-coded microspheres, or beads, are internally dyed with different proportions of red and infrared fluorophores that correspond to a distinct spectral signature, or bead region. The quantification of multiple cytokines and other biomarkers in a sample provides critical information about biological processes and diseases. Multiplex immunoassay Antibodies specific to a desired analyte are coupled to a unique bead region and are incubated with sample. After washing away unbound materials, samples are incubated with a mixture of biotinylated detection antibodies and a streptavidin-phycoerythrin (PE) reporter. Using a Luminex instrument, beads are excited by one laser to determine the bead region and corresponding assigned analyte. Another laser determines the magnitude of the PE-derived signal, which is proportional to the amount of analyte bound. Multiple readings are taken at each bead region, ensuring robust detection https://www.youtube.com/watch?v=AlYOANYnqLk&t=280s Multiplex immunoassay Inflammatory Profile of COVID-19 (+) patients - Patients recently diagnosed with COVID-19 had salivary pro-inflammatory IL-1β and TNF-α increased 1000 IN F C o n c e n tr a tio n ( p g /m L ) 750 IL - 1 0 IL - 1 b 500 IL - 6 IL - 4 200 TNF 100 0 0 -6 -4 F -1 -1 F IL IL IN N IL IL T 2000 SNE C o n c e n tr a tio n ( p g /m L ) 1500 B lo o d S e ru m 1000 500 100 80 60 40 20 0 0 -6 -4 F -1 -1 F IL IL IN N IL IL T Proteomics workflow - Mass spectrometry-based proteomics SEPARATION/PURIFICATION DETECTION/IDENTIFICATION Chromatographer Mass Detector Spectrometer UV-VIS (Absorption) LC-MC Differential refractometer - Refractive index Fluorometric (Florescence) Electrochemical (ECD) (Oxidation reduction) Conductivity Mass spectrometry Proteomics workflow - Mass spectrometry-based proteomics Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Mobile phase - liquid Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography -HPLC HPLC – High Performance Liquid Cromatography Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Separation Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Norma Phase Polar stationary phase and non-polar solvent Reverse Phase Non-polar stationary phase and polar solvent Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Detection/Identification Determination Peak area (or height) is proportional to the concentration (or amount) of the component The concentration of component A (caffeine) is measured by comparing its peak area to the peak area of a standard caffeine reference. Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Detection/Identification Retention time in LC is the time required for a particular molecule to pass through the column. It is a measure of the time taken for a solute to pass through a chromatography column Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Detection/Identification Degree of separation Proteomics workflow - Mass spectrometry-based proteomics Proteomics Liquid Chromatography Advantages of LC Rapid and efficient separations Sensitive, selective and non-destructive detectors Can inject aqueous samples Running cost low Applications of LC Proteomics workflow - Mass spectrometry-based proteomics Proteomics Gas Chromatography Mobile phase – Gas (He or N) and the components are separated in their gas phase Proteomics workflow - Mass spectrometry-based proteomics Proteomics Gas Chromatography It is used to separate and detect small molecular weight compounds in the gas phase. The sample is either a gas or a liquid that is vaporized in the injection port. The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert. The pressure is applied and the mobile phase moves the analyte through the column. The separation is accomplished using a column coated with a stationary phase. Proteomics workflow - Mass spectrometry-based proteomics Proteomics Gas Chromatography Proteomics workflow - Mass spectrometry-based proteomics Chromatographer Mass Spectrometer Mass Spectrometry (MS) Is an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. m/z ratio : Molecular weight / Charge Proteomics workflow - Mass spectrometry-based proteomics Instrumentation and Steps Proteomics workflow - Mass spectrometry-based proteomics Proteomics workflow - Mass spectrometry-based proteomics Appling charge to the molecules Proteomics workflow - Mass spectrometry-based proteomics Appling charge to the molecules Illustration of ionization Ion evaporation MALDI (Matrix Assisted Laser Desorption Ionization) forms singly charged ions Direct measurement of molecular ion mass as [M+H]1+ Electrospray Ionization (ESI) forms multiply charged ions Mass analyser /Detection Mass analyser /Detection ION TRAP MS Spectra – a lot of numbers Mass spectrometry-based proteomics MALDI-TOF mass spectrum of three proteins; ubiquitin, cytochrome C, and equine myoglobin. Figure adapted from King’s College London Mass spectrometry-based proteomics The electrospray ionization process Mass spectrometry-based proteomics Tandam Mass Spectrometry - MS/MS approach Used to measure fragmentation spectra and identify proteins at high speed and accuracy. Mass spectrometry-based proteomics Tandem MS: obtaining amino acid sequence information Mass spectrometry-based proteomics A common nomenclature for sequence ions Mass spectrometry-based proteomics Protein Identification by Peptide Mass Fingerprinting (PMF) Is a high throughput protein identification technique in which an unknown protein is digested with endoprotease to yield the constituent small peptides. The accurate mass of these peptides is determined by MS analysis. This gives the peak list of peptides of the unknown protein. This peak list is compared with the theoretical peptide peak list obtained from the in silico digestion of the database proteins and the best match is identified by computer software. Mass spectrometry-based proteomics Isotope labelling combined with mass spectrometry (MS) can improve sensitivity, selectivity, accuracy, and throughput. There are two main approaches: chemical isotope labeling (CIL) multiplex isotope labeling. These approaches can be used for quantitative proteomics in the data- dependent acquisition mode. iTRAQ reagentes - labelling Mass spectrometry-based proteomics Protein Identification Strategy Problem-Solving Describe the process of protein identification using LC-MS/MS Problem-Solving MOODLE : TWO QUESTIONS Delivery by email: [email protected] Deadline: 17/12/2024