Proteomics: 3rd-Year Biotechnology Department Past Paper (Dec 2024)

Ecole Nationale Supérieure de Biotechnologie Biotechnology Departement « 3rd-year » Proteomics Dr. MADDI FERDJOUKH T t.maddi @ensbiotech.edu.dz  Chapter II Protein Identification and analysis Extraction of Pr...

Ecole Nationale Supérieure de Biotechnologie Biotechnology Departement « 3rd-year » Proteomics Dr. MADDI FERDJOUKH T t.maddi @ensbiotech.edu.dz  Chapter II Protein Identification and analysis Extraction of Proteins Separation Identification Protein visualization and stainingSeparation of Proteins Coomassie blue (triphenylmethane) 3 Protein visualization and stainingSeparation of Proteins Silver nitrate http://www1.ucam.ac.ma/biochimie-genetique-S5/biomolecules-plateforme/TECHNIQUES- Loïc N Michel, 2005 (thesis) ANALYSES/proteomique/proteomique.html 4 Protein visualization and stainingSeparation of Proteins Protein visualization and stainingSeparation of Proteins Coomassie blue (video, link in Moodle) 7  Separation of Proteins Detect specific protein Western Blot Laboratory method that uses antibodies to detect target proteins in a sample. 8  Western Blot Hegyi, G., Kardos, J., Kovács, M., Málnási-Csizmadia, A., Nyitray, L., Pál, G.,... & Venekei, I. (2013). Introduction to Practical Biochemistry. ELTE Faculty of Natural Sciences, Institute of Biology. 9  Western Blot 10  Western Blot 12 Western blot (video, link in Moodle) 14  5- Révélation d/ Western blot Revelation - Résultat: Picture (Spot) analysis Melanie DeCyder SameSpot Delta 2D Artefacts and contamination High Salt Concentration Nucleic acid Liang, X., Wang, J. R., Wong, K. W. V., Hsiao, W. L., Zhou, H., Jiang, Z. H.,... & Liu, L. (2014). Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples. Molecular medicine reports, 9(2), 626-632. Picture (Spot) analysis Mélanie DeCyder SameSpot Delta 2D Spot allignement Digitalisation (numérisation): automatic protein detection Quantification: measuring the grayscale intensity of proteins Matching: a matching algorithm compares gels 2 by 2 to find spots that represent the same protein in both gels  Example : Melanie IV software Gel a (left) and gel b (right) are presented here, each accompanied by a detail of spot detection (circled in red) done manually by an expert. Figure : Gels choisis pour l'étude. Le gel à (gauche) et le gel b (droite) sont présentés ici, accompagnés chacun d’un détail de la détection des spots (entourés en rouge) faite manuellement par un expert. Reproductibility and inter-gel variations Sypro Ruby Table :Some software for analyzing two-dimensional electrophoresis gel images Fabien, 2008 22 2D-DIGE Differential gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three different protein samples can be labeled with size-matched, charge-matched spectrally resolvable fluorescent dyes (for example Cy3, Cy5, Cy2) prior to two dimensional gel electrophoresis. 2D-DIGE This allows precise analysis of differences in abundance of each protein between samples. 24 https://www.abdn.ac.uk/ims/documents/2D-DIGE_Aberdeen_Proteomics.pdf  2D-DIGE Cyanine dyes are used to label proteins, to be used in a variety of fluorescence detection techniques Cyanines, also referred to as tetramethylindo(di)-carbocyanines Les Cyanines Dyes = Cy-Dyes Cy3 – Cy5- Cy2 Samples labeled with fluorescent dyes before separation 25 2D-DIGE http://icim.marseille.inserm.fr/spip.php?article35 26  2D-DIGE Example : cerebrospinal fluid (CSF)= (Liquide LCR) Hayward et al, 2009. Surrogate Markers for Epileptogenesis 27 2D-DIGE 28 https://www.appliedbiomics.com/2d-dige/data-analysis-in-gel/ 29 2D-DIGE Main applications: Identifying therapeutic or diagnostic markers in different diseases Identifying key regulators in major biological pathways Studying post-translational modifications such as phosphorylation, glycosylation, etc. 30  2D-DIGE Two examples of DIGE-based PTM analysis are shown in Fig. Panel “A” shows postsynaptic densities isolated from rat hippocampus (were treated following the protocol). As indicated by the arrows, spots shown as green in the 2D gel correspond to ubiquitinated proteins, which can be removed from the polyacrylamide gels and identified by mass spectrometry. Analysis of Protein Posttranslational Modifi cations Using DIGE-Based31Proteomics from : DeKroon et al,2012 2D-DIGE Panel “B” displays a portion of the 2D gel in which dephosphorylated proteins (protocol in paper) were separated by gel electrophoresis. The absence of the phosphate group (or groups) induces pI shifts (lower portion of the figure) that can be used to determine the presence of the phosphate group in the untreated (phosphorylated) sample Analysis of Protein Posttranslational Modifi cations Using DIGE-Based32Proteomics from : DeKroon et al,2012 2D-DIGE 33 https://www.appliedbiomics.com/2d-dige/data-analysis-in-gel/ 34 2D-DIGE 35  2D-DIGE Figure : Comparison of mouse liver protein visualised by different fluorescence 36  2D-DIGE 37 http://icim.marseille.inserm.fr/spip.php?article35  2D-DIGE/ 2D PAGE 2D DIGE 2D PAGE Revelation Cydye Coloration/fluorescence - 50ng/spot (coomassie blue) Sensitivity/ spot 0,2ng/spot - 1ng/spot (Silver nitrate and Sypro Ruby) Sample number 3 per gel 1 per gel Comparison number 3 in 1 gel 1 in 2 gels Spot Resolution High Low Reproducibility High Low 38 www.appliedbiomics.com  Excision Proteins extracted from SDS-PAGE gel 39 Brunelle et Green 2014  Excision Proteins extracted from SDS-PAGE gel Fig: The proteins extracted from SDS-PAGE gels were subjected to re-electrophoresis and quantitated by image analysis 40 Jin et Manabe , 2005 Gel destaining The gel will be destained by adding Coomassie blue : ammonium bicarbonate/acetonitrile and vortexing Nitrate d’argent : hydrogen peroxide (H2O2) + sodium thiosulphate This is done by using a destaining solution, which takes approximately 10 minutes to overnight to remove excess stain and produce bands with clear background. 41 Extraction (other reagents used for extraction) from tissues, cells, biological fluids… Goal: extract the maximum amount (concentration)of protein Buffer  Organic acids, which damage membranes and solubilize membrane proteins (e.g. formic acid (FA) and trifluoroacetic acid (TFA))  Organic solvent-water mixtures, which facilitate membrane rupture and denature proteins (e.g. acetonitrile-water and methanol-water) Centrifugation : analysis of the supernatant (A. E. Speers et al., 2007). 42 Purification A pretreatment step can eliminate contaminants and/or detergents : Sample purification or removal of contaminants (lipids, salts, etc.) can be carried out by protein precipitation (trichloroacetic acid (TCA), acetone, chloroform-methanol mixture or ethyl acetate) Channaveerappa et al., 2019 43 Purification This step is necessary to limit signal suppression and interference and thus facilitate protein identification.Otherwise repeat an SDS page (M. Chevallet et al., 2007). 44  Hydrolyse enzymatique des protéines Chaotrope agents (Trifluoroethanol 5 %, urea/Thiourea 5/2 M) Denaturation/ break down S-S bridge Detergents: dithiothreitol (DTT) (between 4mM et 300mM) Alkylation : using iodoaceamide –IAA (50 à 300 mM) , an alkylating agent to avoid the reformation s_s Digestion: following mass ratio are used : enzyme/protein = 1/50 (1 μg of protein/0.02µg E). 6.digestion https://tel.archives-ouvertes.fr/tel-03636477/document 45 Digestion by using trypsine NH2-XXXXXXKXXXXXXRXXXXX-COOH NH2-XXXXXXK-COOH NH2-XXXXXXR-COOH NH2-XXXXXX- COOH 46  identification of Proteins Protein identification and validation steps Zhang et al 2007, MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid 47 proteomes  identification of Proteins identification of Proteins MASS SPECTROMETRY Zhang et al 2007, MAPU: Max-Planck Unified database of organellar, cellular, tissue and 48 body fluid proteomes  identification of Proteins MASS SPECTROMETRY (INSTRUMENT) Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition 49  identification of Proteins MASS SPECTROMETRY A mass spectrometer is an analytical instrument used to measure the exact molecular mass of a sample by breaking the initial molecule into smaller, charged fragments. This could be used either to identify an unknown compound or validate the product of a synthetic process, by calculating the molecule's mass to charge ratio. 50  identification of Proteins MASS SPECTROMETRY Mass spectrometry (MS) is a highly effective qualitative and quantitative analytical technique used to identify and quantify a wide range of clinically relevant analytes. When coupled with gas or liquid chromatographs, mass spectrometers expand analytical capabilities to various clinical applications. In addition, due to its ability to identify and quantify proteins, mass spectrometry is an essential analytical tool in the field of proteomics. 51 Garg et al, 2023  identification of Proteins MASS SPECTROMETRY Mass spectrometry purpose?  Measures the mass-to-charge ratio (m/z) of ions to identify and quantify molecules in simple and complex mixtures. Identify unknown compounds via molecular weight determination  Protein sequencing Spake protein - Virus SARS CoV-2 52  identification of Proteins MASS SPECTROMETRY 53  identification of Proteins MASS SPECTROMETRY COMPONENTS The three fundamental parts of a mass spectrometer are the ionization source, the mass analyzer, and the detector. https://theory.labster.com/mass-spectrometer/ 54  identification of Proteins MASS SPECTROMETRY COMPONENTS https://theory.labster.com/mass-spectrometer/ 55  identification of Proteins MASS SPECTROMETRY COMPONENTS 56  identification of Proteins MASS SPECTROMETRY IONIZATION (ION SOURCE) Ionization Source: Once the sample has been loaded into the instrument, it is turned into a gas and charged with electric and magnetic fields: this is called Electrospray Ionization (ESI). An alternative is the MALDI technique, where the reagent is crystallized and then ionized by using a laser. 57  identification of Proteins MASS SPECTROMETRY IONIZATION (ION SOURCE) Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB) Biomolecules Field Desorption / Field Ionisation (FD/FI) Matrix Assisted Laser Desorption Ionisation (MALDI) Thermospray Ionisation (TSP) 58  identification of Proteins IONIZATION (ION SOURCE) Ionization source Peptides are ionized directly at the column outlet MALDI (Matrix Assisted Laser Desorption Ionisation) ESI (Electrospray Ionization) Allows the formation of ions by excitation of a matrix produce ions using an electrospray in which a high voltage containing the crystallized analyte is applied to a liquid to create an aerosol. The matrix consists of crystallized molecules formation of ions by nebulization of the analyte 59  identification of Proteins IONIZATION (ION SOURCE) Spectrométrie de Masse (MS) ESI-MS Electrospray : ElectroSpray Ionisation (ESI) Ionization Mass Spectrometry Electro nebulization 60  Electrospray Ionisation (ESI) IONIZATION (ION SOURCE) The transfer of ionic species from solution into the gas phase by ESI involves three steps: (1) dispersal of a fine spray of charge droplets, followed by (2) solvent evaporation and (3) ion ejection from the highly charged droplets. tube, which is maintained at a high voltage (e.g. 2.5 – 6.0 kV) relative to the wall of the surrounding chamber. Ho et al , 2003 61  identification of Proteins IONIZATION (ION SOURCE) Electrospray Ionisation (ESI) figure 1 62 http://www.chm.bris.ac.uk/ms/esi-ionisation.xhtml  Electrospray Ionisation (ESI/ MS) figure 2 63 http://www.chm.bris.ac.uk/ms/esi-ionisation.xhtml  Electrospray Ionisation (ESI) 64  Electrospray Ionisation (ESI) Ho et al , 2003 A single protein will show its characteristic cluster ions 65 containing multiple-charged ions. Protéomique Electrospray Ionisation (ESI) Spectrométrie de Masse (MS) ESI-MS : ElectroSpray Ionization Mass Spectrometry NH2 + O + NH2 O OH O H N OH O N N H H N O N O O OH O H NH2 N OH O NH NH2 + N H N H N O N O O + NH2 O OH O O NH + H N OH N N H H N O N O + NH2 Le même peptide peut avoir O NH différents états de charge 66  ESI-MS : ElectroSpray Ionization Mass Spectrometry We are detecting protein molecules to which additional Calcul de la masse + H atoms are attached. The m/z of a particular peak is: - m/z = (PM + nH+)/n m/z = [PM + nH+] /n m/z = [PM + (n+1)H+] /(n+1) +1 H+ -PM: poids moléculaire -n: nombre de charges -H+: masse d’un proton 67 M or PW? 1431.6 = (M + nH+)/n et 1301.4 = *M + (n+1)H+] /(n+1) n(1431.6) - nH+ = (n+1)1301.4 - (n+1)H+ n(1431.6) = n(1301.4) +1301.4 - H+ n(1431.6 - 1301.4) = 1301.4 - H+ M n = (1301.4 - H+) / (1431.6 - 1301.4) m/z 1431.6 = 1300.4/130.2 = 10 1431.6 = (PM + nH+) n donne 1431.6 x 10 = PM + (10 x 1.008) PM = 14,316 - 10.08 PM = 14,305.9 Da 68 Theorical Exemple : M = 1000 kda 69  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra 70  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra 71  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra M= MW 72  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra 73  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra 74  Electrospray Ionisation (ESI) Example : Horse myoglobin protein ESI MS spectra 75  Electrospray Ionisation (ESI) Le logiciel de déconvolution permet, par un procédé mathématique, d’extraire un signal d’une matrice complexe afin d’obtenir des spectres de masse épurés Deconvolution (AMDIS software) Ashcroft, A. E. (2011). An introduction to mass spectrometry. University of Leeds. 76  Electrospray Ionisation (ESI) ESI-MS Spectra m/z of a protein Analysed adding water Presence of acetonitrile solution +formic acid Ashcroft, A. E. (2011). An introduction to mass spectrometry. University of Leeds. Chapitre 2 : Analyse protéomique 77  identification of Proteins IONIZATION (ION SOURCE) MALDI MALDI techniques typically employ the use of UV lasers (337 nm) Matrix Protect the sample: the matrix absorbs the laser energy and then transfers it to the sample to avoid direct laser irradiation of the sample and lead to decomposition of the sample molecules. 78  identification of Proteins IONIZATION (ION SOURCE) MALDI The MALDI method is divided into three steps. First, the sample is mixed with the appropriate substrate material and applied to the metal plate. Secondly, the pulse laser irradiates the samples, triggering (déclenchant) the ablation and desorption of samples and matrix materials. Finally, the analyte molecules are ionized by protonation, and then accelerated to the mass analyzer to analyze them. 79  identification of Proteins In a tiny region, in a very short time interval (on the order of IONIZATION (ION SOURCE) ns), the laser provides high-intensity pulsed energy to the analyte on the target, allowing it to desorb and ionize in an MALDI instant 80  identification of Proteins IONIZATION (ION SOURCE) MALDI 81  identification of Proteins IONIZATION (ION SOURCE) MALDI 82 MALDI Laser Analyte (M+H)+ + Désorption + + Analyte (M) Matrice Compared with other mass spectrometry ionization techniques such as electron bombardment ionization and chemical ionization, MALDI technology has the following characteristics: It can ionize some samples that are difficult to ionize by other ionization techniques (especially biomacromolecules), and obtain complete ionization products without obvious fragments; It has a dominant single-charged molecular ion peak, and simple mass spectrum, which is suitable for analysis of multi-component samples; 83  identification of Proteins IONIZATION (ION SOURCE) MALDI matrix Use of solid (crystalline) organic matrices 84  Spectrométrie de Masse (MS) identification Le MALDI TOF of Proteins IONIZATION- Matrix-assisted (ION SOURCE) laser desorption/ionization Time of Flight (temps de vol) MALDI matrix Dépôt sur la plaque + Co-cristallisation 85  identification of Proteins IONIZATION (ION SOURCE) MALDI The choice of matrix is one of the most important steps in MALDI analysis. The ideal matrix generally has the following properties: 1. strong electron absorption at the laser wavelength used; 2. Better vacuum stability, lower vapor pressure, and good miscibility with the analyte in the solid state. 86 http://www.perrin33.com/biochanalys/ms_2.php  identification of Proteins IONIZATION (ION SOURCE) MALDI In MALDI technology, the matrix has a special effect on the analysis of the sample: dilute the sample to dissociate the clustered macromolecule; protect the sample, the matrix absorbs the laser energy and then transfers it to the sample to avoid direct laser irradiation of the sample and lead to decomposition of the sample molecules. The choice of matrix depends primarily on the wavelength of the laser used, followed by the nature of the object being analyzed. 87 http://www.perrin33.com/biochanalys/ms_2.php WHICH IONIZATION SOURCE? MALDI ESI 2 D PAGE HPLC Choice depending on molecule size Sample Volume 88 https://tel.archives-ouvertes.fr/tel-00625749/document MALDI  identification of Proteins To identify a protein, it should be necessary to digest it with trypsin and analyze it with MALDI MALDI-TOF, then …analysis with dedicated software (MASCOT) Zhang et al 2007, MAPU: Max-Planck Unified database of organellar, cellular, tissue and 91 body fluid proteomes  Spectrométrie de Masse (MS) Mascot Le MALDI TOF Peptide Masse fingerprinting (PMF) Protéine identifiée VAN DORSSELAER, A. (2006). Nouvelles méthodologies dans l’analyse protéomique par spectrométrie de masse. Application à la recherche 92 de biomarqueurs dans le cadre des leucémies. Analyzers A mass analyzer is the component of the mass spectrometer that takes ionized masses and separates Types of Analyzers them based on charge to mass ratios and outputs them to the detector where they are detected and later Quadrupole (Q) converted to a digital output Time-of-flight (TOF) Résolution : exemple: un appareil permettant de distinguer les ions 100 et 100,1 possède une résolution de 100/0,1=1000 Magnetic Sector Résolution = m/Δm Fourrier transformer Ion Trap 93 Analyzers Quadrupole (Q) Les barres sont soumises à un potentiel électrique. Cela va faire osciller les ions traversant la zone comprise entre les barres Pierre Dubreuil. Introduction à la SPECTROMÉTRIE DE MASSE. 94 www.rocler.qc.ca Analyseurs The principle of ToF mass analyzer involves the separation of ions based on the time it takes for the ions to travel through a TOF (Time of Flight) , Temps de vol flight tube with known length and reach the detector. L'analyseur à temps de vol consiste à mesurer le temps que met un ion, accéléré préalablement par une tension, à parcourir une distance donnée. Le rapport masse sur charge est directement mesurable à partir du temps de vol. 95 MALDI- TOF MS et ESI-TOF MS les molécules ionisées sont accélérées puis transférées dans l'analyseur TOF. Cet analyseur va permettre la séparation des molécules ionisées qui dépendra de leur rapport masse sur charge (m/z). 96 Tandem mass spectrometry MS/MS or MS2 It consists of “breaking” a molecule inside a mass spectrometer, in order to determine its structural properties How? : by using several analyzers arranged one after another. This system will increase the precision of analyses 97 Tandem mass spectrometry MS/MS or MS2 98 Tandem mass spectrometry MS/MS or MS2 99 Tandem mass spectrometry MS/MS or MS2 Lopez, F. (2005). Protéomique et spectrométrie de masse: applications en clinique. Hépato-Gastro & Oncologie Digestive, 12(6), 427-435. 100 De novo sequencing using mass spectrometry In mass spectrometry, de novo peptide sequencing is the method in which a peptide amino acid sequence is determined from tandem mass spectrometry Knowing the amino acid sequence of peptides from a protein digest is essential for studying the biological function of the protein. De novo sequencing using mass spectrometry Mass spectrometry sequencing refers to the determination of the amino acid sequence of the primary structure of a sample based on mass spectrometry De novo sequencing using mass spectrometry Peptide fragmentation in MS-based de novo sequencing. (a) An illustrative fragmentation Greff et al 2022 spectrum. De novo sequencing using mass spectrometry In 1984 , following Roepstorff et Fohlman,: les ions sont formés et la charge est sur différent fragments (N-terminal et C- terminal) sont appelés a, b et c ou bien x, y et z.  De novo sequencing using mass spectrometry La fragmentation en MS/MS va couper le peptide à différents endroits In 1984 , following Roepstorff et Fohlman,: les ions sont formés et la charge est sur différent fragments (N-terminal et C-terminal) sont appelés a, b et c ou bien x, y et z. Nomenclature de fragments peptidiques De novo sequencing using mass spectrometry De novo sequencing using mass spectrometry Novak et al.2009. An application of the metric access methods to the mass spectrometry data De novo sequencing using mass spectrometry Example De novo sequencing using mass spectrometry Find Aa 7 Find Aa 7 Find Amino acid 7 Molecular Weights of the Amino Acids Example 2 Example 2 Example 2 ion y (trypsin digestion) Example 2 ion y (trypsin digestion) Modifications post-traductionnelles les plus communes et les plus importantes (incrément)  Identification identification des protéines of Proteins Stratégie TYPES OF PROTEOMICS bottom-up WORKFLOWS bottom-up strategy VAN DORSSELAER, A. (2006). Nouvelles méthodologies dans l’analyse protéomique par spectrométrie de masse. Application à la recherche deDORSSELAER, VAN biomarqueurs dans leNouvelles A. (2006). cadre des leucémies. dans l’analyse protéomique par spectrométrie de masse. Application à la recherche méthodologies 120 de biomarqueurs dans le cadre des leucémies.  identification of Proteins TYPES OF PROTEOMICS WORKFLOWS bottom-up strategy Peptide Masse fingerprinting (PMF) VAN DORSSELAER, A. (2006). Nouvelles méthodologies dans l’analyse protéomique par spectrométrie de masse. Application à la recherche 121 de biomarqueurs dans le cadre des leucémies.  identification of Proteins TYPES OF PROTEOMICS WORKFLOWS Peptide Masse fingerprinting (PMF) bottom-up strategy VAN DORSSELAER, A. (2006). Nouvelles méthodologies dans l’analyse protéomique par spectrométrie de masse. Application à la recherche 122 de biomarqueurs dans le cadre des leucémies. MASCOT Mascot est un moteur de recherche logiciel qui utilise des données de spectrométrie de masse pour identifier des protéines à partir de bases de données de séquences peptidiques Figure: Illustration de la démarche d’une analyse classique en protéomique. Source : University of Oslo, http://www.biotek.uio.no 126  Spectrométrie de Masse (MS) Mascot Le MALDI TOF Peptide Masse fingerprinting (PMF) Protéine identifiée VAN DORSSELAER, A. (2006). Nouvelles méthodologies dans l’analyse protéomique par spectrométrie de masse. Application à la recherche 127 de biomarqueurs dans le cadre des leucémies.  128 http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF Protein database Universal Protein knowledgebase (UniProt) consists of two parts: UniProt/SwissProt : garantit une validation des données par des experts Uniprot/TrEMBL qui contient les traductions automatiques des séquences génomiques sans expertise complémentaire associée. 129 Protein database Figure : Évolution du nombre d’entrées dans UniProt/TrEMBL ( durant 15 années ) Source : European Bioinformatics Institute (EBI), http://www.ebi.ac.uk/uniprot/TrEMBLstats/ 130