L13-Proteomics PDF

§ Design of innovative experiments Gregor Mendel, Hershey-Chase, Meselson-Stahl, etc. § Fundamental discoveries DNA as the genetic material, central dogma, genetic codon, etc. § Evolution: how life (living systems) came about § Engineering principles Feedback regulation, trp and lac...

§ Design of innovative experiments Gregor Mendel, Hershey-Chase, Meselson-Stahl, etc. § Fundamental discoveries DNA as the genetic material, central dogma, genetic codon, etc. § Evolution: how life (living systems) came about § Engineering principles Feedback regulation, trp and lac operons, sensing by Hb, etc.  Properties that “emerge” out of an orderly arrangement or interactions of a set of components ENDOPLASMIC RETICULUM (ER) Nuclear envelope Rough ER Smooth ER Nucleolus NUCLEUS Flagellum Chromatin Centrosome Plasma membrane CYTOSKELETON: Microfilaments Intermediate filaments Microtubules Ribosomes Microvilli Golgi apparatus Peroxisome Mitochondrion Lysosome cutaway view of a generalized cell Figure 6.8a in Biology. A global approach by Campbell et al., Global Ed.  Output (or input) per unit time 2,000 chapatis per hour https://www.rotipro.in/product/softika-majestic- 2000/?gad_source=1&gclid=Cj0KCQjw0Oq2BhCCARIsAA5hubXcecSqRBU5twgzJUF0Xaf50GBqzVf3INlsvv9kaxjdo3tPr8GQE7AaAkSPEALw_wcB https://www.freepik.com/free-photo/bakery_3545079.htm#fromView=search&page=1&position=48&uuid=d1c37bae-eb06-4461-9833-2476f0e89ce5  Suffix Meaning -ome The complete set -omics The study of an -ome High-throughput techniques are key “tools” in our efforts to study Biology at the cellular, tissue, and organism levels § Study of the proteome of a cell Proteome: all proteins present in a cell § Proteome is dynamic, not static Varies as a function of time (temporal changes) Varies from one cell type to another (spatial changes) Varies within different compartments of a cell (spatial changes) § -omics are complementary to one-protein-at-a-time type of studies Type of information obtained is different Types of questions that one poses are also different § Relate changes in the proteome to “functional” state How does the proteome change when a normal cell become malignant? § Information about changes in the proteome can provide clues... Cannot provide answers to questions such as the one posed above § Help in identifying biomarkers Useful for diagnostics https://media.istockphoto.com/id/1197802707/photo/kerman-kerman-province-iran-a-huge-variety-of-spices-and-herbs-on-a-counter-on-a- traditional.jpg?s=1024x1024&w=is&k=20&c=sYMnQOsqTfAYxb7jX-pu1E7NZq5floGKUROT3MY_TJI=  Do chefs use all ingredients for all the dishes? Table salt Wheat flour Cardamom (elaichi)... https://cdn.pixabay.com/photo/2023/08/21/15/30/chef-8204538_1280.png § GENOME: the entire genetic material of an organism § GENOMICS: the study of the genome of an organism Example: determining and analyzing the DNA sequence 5 µm Figure 13.3. Campbell’s Biology, 10th ed. § Every one of the trillion cells have the same genome (i.e., DNA) § But will they make + use the same set of proteins? § Every one of the trillion cells have the same genome (i.e., DNA) § But will they make + use the same set of proteins? § Each cell synthesizes a different set of proteins Depends upon its functional requirement § Proteins perform a range of cellular functions § Disease is the result of protein malfunction § Drugs either alter protein function or are proteins themselves Butterfly Frog Campbell, 10th Edition § Caterpillar and butterfly share same genome; & tadpole and the frog share same genome but their proteome is very different, which accounts for the variation. § Genome is static but proteome is dynamic and therefore, an organism’s phenotype is determined by the proteome. § The dynamic proteome, not the static genome, determines an organism’s phenotype.  Protein PTMs Hydroxylation OH Phosphorylation P P OH Methylation OH P Glycosylation CH3 OH P Glucose Glucose CH3 CH3 Glucose Glucose CH3 Many proteins undergo PTM at some of their amino acid residues after synthesis process. Hydroxylation, methylation, alkylation, acylation are commonly observed modifications. § Every one of the trillion cells have the same genome (i.e., DNA) § But will they make + use the same set of proteins? § Each cell synthesizes a different set of proteins Depends upon its functional requirement § Even for the same cell type, the set of proteins that it has depends upon metabolic requirements Proteins are made when needed; degraded when the requirement ceases § Spatiotemporal expression of genes Spatial = cell type; temporal = time (external conditions) § The study of the entire set of proteins i.e., the proteome for a given cell type under the specified growth (or metabolic) conditions § What are studied? Which proteins are made (regulation of gene expression) How much of each protein is made (level of expression) Where is a protein present (subcellular localization) § It is possible that some proteins are made in higher (or lower) amounts Upregulation and downregulation of gene expression  GCCTTTAAGGATCCGGA TTGCAAATCCCGATTCG GGCGTTATGGCTTGGAA CCCGGATGCCTGGTCCA DATABASE SEARCH – Corresponding protein sequence obtained from genomic sequences Available genome database MS sequencing Ala-Leu-Val-Cys-Trp-Tyr-Ala-Gly-Gly-Tyr-His- Pro-Met-Arg-Ile-Lys-Lys-Glu-Ser-Pro-Thr-Thr- Val-Val-Gln Protein Protein sequence Genome databases help in identifying gene sequence of a protein that has been sequenced by mass spectrometry. § Suffixes –ome and –omics Genome is the entire DNA of an organism Genomics is the study of the genome of an organism Every cell in an organism will have the same genome § Proteome is the entire set of proteins of a cell It is essential to study proteome for different cell types and under different metabolic, growth, disease,... conditions  o r io n Ne dific tein tw at ks M Pro Int Pro o Ne era tein tw ctio or n ks DYNAMIC PROTEOME Genome Metabolome Environmental Transcriptome Factors COMPREHENSIVE VIEW OF BIOLOGICAL PROCESSES  Extraction of all proteins from cells Separation of proteins (in 2-dimensions) Identification of each and every protein spot Characterization https://skin.science/mass-spectrometry https://www.labxchange.org/library/pathway/lx-pathway:bedd0ea1-4d01-40c9-8a4f-23d88d2ae9e9/items/lb:LabXchange:7b3781ab:lx_image:1/56498 § To individually separate from each other a set of proteins present in a mixture § The earliest reported attempts were in the 1950s § There have been several technological advancements and intellectual inputs § Present day technologies are able to separate and identify nearly 10,000 proteins from each other Smithies, O. and Poulik, M. D. (1956) Two-dimensional gel electrophoresis of serum proteins. Nature, 177, p1033. Discovery Proteomics Gel-based, Mass Spec Functional Proteomics Protein chips, Biosensors High Throughput  1. Gel Electrophoresis a. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis): Purpose: Separates proteins based on molecular weight. How it works: Proteins are denatured with SDS (a detergent), which gives them a uniform negative charge. When an electric field is applied, proteins migrate through a polyacrylamide gel, with smaller proteins moving faster than larger ones. Application: Commonly used to estimate protein size, purity, and composition. b. 2D Gel Electrophoresis (2D-PAGE): Purpose: Separates proteins based on two properties—isoelectric point (pI) and molecular weight. How it works: In the first dimension, proteins are separated by their pI using isoelectric focusing (IEF). In the second dimension, they are separated by size using SDS-PAGE. Application: Useful for studying complex protein mixtures and identifying isoforms or post-translational modifications § Electrophoresis: the phenomenon wherein a molecule with a net charge moves in an electric field § Electrophoretic separations are carried out in gels which serve as molecular sieves Mobility is related to the size/shape of a molecule relative to pore size Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p71-72 Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p71-72 Side chains of some amino acids are ionizable The pH at which they ionize varies (chemical structure, microenvironment) Figure 5.15. Campbell’s Biology, 10th ed. p77 § Isoelectric point (pI) of a protein = pH at which its net charge is zero § Mobility of a protein in an electric field at pI is zero When pH < pI, net charge > 0 When pH > pI, net charge < 0 § Cytochrome c: pI = 10.6 § Serum albumin: pI = 4.8 Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p73-74 Start of the experiment End of the experiment Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p73-74 Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p73-74  Each spot corresponds to a protein or a group of proteins which have near identical net charge, MW, and shape Section 3.1, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p73-74  Electrical Engineer Analytical Chemist § Technique for analysing ionized forms of molecules § Mass measurements are obtained by determining how readily an ion is accelerated in an applied electric field § Sample amount: picomoles (pmol) to femtomoles (fmol) § However, mass alone is inadequate to identify a protein Section 3.5, Biochemistry by Berg, Tymoczko, Stryer (6th ed.), p93 onwards  Ionization Mass Analyzer Detection Ion Source Mass Analyzer Ion Detector Detects ions Forms ions (charged molecules) Sort Ions by Mass (m/z) Data System Data Processing Mass Spectrum Abundance Sample Relative Inlet 1000 2000 m/z § 2D gel electrophoresis → Treat the gel with a protease e.g., trypsin Trypsin cleaves only certain peptide bonds (C=O group from Lys, Arg) § Determine the masses of the fragments § Match peptide masses against proteins in a database  Short-finned pilot whale Hippopotamus Evolution Paleontology Species interactions Figure 5.26 of Campbell Biology by Medical science Conservation Reece and others (10th edition) § A cancer of the bone marrow and the blood that progresses rapidly without treatment § Diagnosis and progression of AML is based on morphologic, immunophenotypic, cytogenetic and molecular assessments § Need for new markers for diagnosis and prognosis Journal of Proteomics 303 (2024) § Several proteins were found to be differentially expressed Use this data to understand disease biology § Potential marker proteins: pro- platelet basic protein (CXCL7), enolase 1 (ENO1) and beta-2-glycoprotein 1 (APOH) § Much larger clinical cohorts is required to establish the utility as a marker Devise sensors to detect one or more of these proteins from a small volume of blood or bone marrow interstitial fluid Community of microbes that co-exist and cooperate with each other Multi-species oral biofilm Sci. Rep. (2016) 6:27537 § Used proteomics to investigate protein abundances in community and single species biofilms Changes occurring in active metabolic pathways due to inter-species interactions § Community development is dependent on cooperative interactions Competition for limited resources was also found How does the system evolve as a consequence of cooperation and competition? § Several pathways are involved in biofilm formation in mixed communities Sci. Rep. (2017) 7:16483 § Human Proteome Project will generate a map of the protein-based molecular architecture of the human body become a resource to help elucidate biological and molecular function advance diagnosis and treatment of diseases https://www.youtube.com/watch?v=85Ou_kotABk  Phenome Metabolome Proteome Transcriptome Genome Figure 21.4, Campbell