LO9 Protein Identification and Analysis (1) PDF

Bio16 Computational Biology Protein Identification and Analysis Prepared by: Joseph Martin Q. Paet Biology Department, College of Science...

Bio16 Computational Biology Protein Identification and Analysis Prepared by: Joseph Martin Q. Paet Biology Department, College of Science Bicol University 1 Proteins polypeptide polymers consisting of a linear arrangement of amino acids Have diverse function Structural (e.g., actin contributes to the cytoskeleton) Enzymatic (e.g., hexokinase as the first catalyst of glycolysis) Transport (e.g., GLUT1 as glucose transporters) Similar to nucleic acids, amino acid sequences can be searched and aligned in databases and analyzed for phylogenetic trees Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 2 1  Mass Spectrometry and Proteomics Proteomics = large-scale determination of gene and cellular function directly at the protein level Mass Spectrometry = method of choice for proteomics ion source + mass analyser =mass- to-charge ratio (m/z) of the ionized analytes detector that registers the number of ions at each m/z value Aebersold and Mann (2003). Mass spectrometry-based proteomics. Nature 422, 198–207 3 Renaissance of Classic Techniques Edman degradation = entails chemical modification of the N-terminal AA, cleavage of this AA from the peptide and determination of the identity of the cleaved labeled AA using HPLC Edman + Fluorosequencing = Massively Parallel Edman Degradation uses single-molecule microscopy and stable synthetic fluorophore chemistry/probes Alfaro, et al. (2021). The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 4 2  Renaissance of Classic Techniques Single-molecule mass spectrometry = can be used to sequence single proteins through MS with a nanopore ion source delivering individual AA directly into the high-vacuum gas phase, where ions can be efficiently detected by their mass- to-charge ratios Alfaro, et al. (2021). The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 5 DNA-facilitated protein sequencing qPAINT Fingerprinting DNA-PAINT with anchor DNA proximity recording FRET-X Structural Fingerprint DNA-PAINT = DNA-based Point Accumulation for Imaging in Nanoscale Topography Relies on the transient binding of dye-labeled DNA strands (imagers) to their complementary target sequence (docking site) attached to a molecule of interest Alfaro, et al. (2021). The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 6 3  Strategies for Nanopore-based Sequencing Biological and solid-state nanopores = give time- dependent and sequence- specific electrical signals Directions a. single-file threading and direct sensing of the sequence b. sensing unique fingerprints in linearized proteins c. identification of folded proteins Alfaro, et al. (2021). The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 7 Emerging Landscape of Protein Sequencing Alfaro, et al. (2021). The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 8 4  Four Perspectives of Protein Analysis Families (Domains & Motifs) Physical Properties Localization Function Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 9 Domains and Motifs: Modular Nature of Proteins Significant structural features and/or sequence identity = signatures, domains, modules, modular elements, folds, motifs, patterns, or repeats Signature = a broad term that denotes a protein category (domain or family or motif) DOMAIN (aka modules) = is a region of a protein that can adopt a particular three‐dimensional structure (fold) MOTIFS (aka fingerprints) = are short, conserved regions of proteins that typically characterize a protein family Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 10 5  Domains and Motifs: Modular Nature of Proteins Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 11 Domains and Motifs: Modular Nature of Proteins Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 12 6  Domains and Motifs: Modular Nature of Proteins Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 13 Many proteins have multiple copies of distinct domains. The most common domain in humans is the immunoglobulin (Ig) domain, and the fibronectin repeat also commonly occurs. These domains are especially prevalent in the extracellular regions of proteins. Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 14 7  Proteins can share a common Lipocalin family domain in a number of ways (a) A domain may essentially extend across the length of a protein. (b) Domains may contain highly Transcriptional regulators related stretches of amino acids that form only a subset of each protein’s sequence. (c)A domain may be repeated Fibronectin III‐like repeat within a single protein (sometimes with many copies). Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 15 Physical Properties of Proteins Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 16 8  Physical Properties of Proteins A variety of post‐translational modifications are added to proteins. Proteintech Group, Inc. (2023, July 13). Post Translational Modifications: An Overview. https://www.ptglab.com/news/blog/post-translational-modifications-an-overview/ Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 17 Gene Ontology Consortium Ontology = description of concepts GO Consortium = a project that compiles a dynamic, controlled vocabulary of terms related to different aspects of genes and gene products (proteins) Represents an ongoing, cooperative effort to unify the way genes and gene products are described 3 main organizing principles of GO Molecular function = the tasks performed by individual gene products Biological process = associated broad goals of a gene product (protein) Cellular compartment = the subcellular localization of a protein Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 18 9 19 20 10  Protein Localization Destiny of protein during or after translation Secreted outside the cell or transported in other compartments Inserted in ER or the plasma membrane (signal recognition particle) Categories based on their relationship to the plasma membrane Soluble = exists within PM, lumen of organelle, or extracellularly Membrane attached = direct/indirect association with PM Targeting depends on motifs KDEL = (Lys, Asp, Glu, Leu) at C-terminus means retention in ER A simplified “road map” of protein traffic within a eukaryotic cell Alberts, B. et al. (2022). Molecular Biology of the Cell (Seventh Edition). W. W. Norton and Company. Canada. Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 21 Protein Function protein is a gene product that interacts with the cellular environment in some way to promote the cell’s growth and function Perspective on Protein Function 1. Biochemical function synonymous with its molecular function Enzymatic, structural, transport, etc. 2. Functional assignment is often made based upon homology Provisional/hypothetical assignment of function 3. Functions may be assigned based on the structure Related 3D folding may signify a similar function 4. All proteins function in the context of their interaction with other molecules Ligands to receptors; substrates to enzymes, etc 5. Many proteins function as part of a distinct biochemical pathway 6. Proteins function as part of some broad cell biological process 7. Protein function can be considered in the context of the proteome Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. 22 11  Bio16 Computational Biology Protein Identification and Analysis References: Aebersold, R., Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003). https://doi.org/10.1038/nature01511 Alberts, B. et al. (2022). Molecular Biology of the Cell (Seventh Edition). W. W. Norton and Company. Canada. Alfaro, J.A., Bohländer, P., Dai, M. et al. The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18, 604–617 (2021). https://doi.org/10.1038/s41592-021-01143-1 Pevsner, J. (2015). Bioinformatics and Functional Genomics (3rd ed.). John Wiley & Sons Inc. Prepared by: Joseph Martin Q. Paet Biology Department, College of Science Bicol University 23 12