Genetics & Genomics

• What is proteomics?

  Proteomics is the genome-wide study of the structure, expression, and interaction of proteins.
• Who coined the term "proteomics" and when?

  The term "proteomics" was coined in the 1990s by joining protein and genomics.
• How does Mark Wilkings define proteomics?

  Mark Wilkings defines proteomics as the study of proteins, their modifications, expression, involvement in metabolic pathways, and interactions.
• What is a proteome?

  A proteome is the set of proteins expressed in a given type of cell at a given time under defined conditions.
• What makes proteomics more challenging than sequencing the genome?

  Proteomics is more challenging due to the dynamic nature of proteins and their high sample complexity.
• How many protein forms are possibly encoded by the human genome?

  There are possibly 100,000 protein forms encoded by approximately 20,235 genes of the human genome.
• What are the learning objectives of the study material?

  Describe post-translational modifications, protein-protein interactions, and core principles of metabolomics.
• Why are proteins considered drug targets?

  Proteins are drug targets because differences in protein expression or post-translational modifications may denote a disease.
• What are post-translational modifications (PTMs) and their roles in the cell?

  • Additions made to proteins after translation
  • Catalyzed by enzymes
  • Important for cell signaling, structure, DNA modification, and protein degradation
• What is phosphorylation in the context of proteins?

  Phosphorylation is the modification of proteins by changing the phosphorylation status of certain amino acids.
• What enzymes are involved in phosphorylation and dephosphorylation?

  Kinases phosphorylate, while phosphatases dephosphorylate.
• Why is phosphorylation targeted as a drug target?

  Because it plays a crucial role in various diseases, such as chronic myelogenous leukaemia.
• How many kinases are present in the human genome?

  There are 518 kinases in the human genome.
• What are the challenges of studying genomics versus proteomics?

  • Genomics:
  • Static (no change with time)
  • Can be amplified (PCR)
  • Low sample complexity
  • Good solubility
  • Proteomics:
  • Dynamic (highly variable with time)
  • Cannot be amplified
  • High sample complexity
  • Various solubility issues
• What percentage of the time is serine used for phosphorylation?

  Serine is used 85% of the time for phosphorylation.
• How many possible phosphorylation sites are there in a eukaryotic cell?

  There are approximately 1,000,000 possible phosphorylation sites in a eukaryotic cell.
• What is the first step in studying the global proteome?

  Define your biological question.
• How does mass-spectrometry-based proteomics help study post-translational modifications?

  It detects characteristic shifts in mass due to PTMs, allowing localization with single amino acid resolution.
• What is the significance of studying the phospho-proteome during SARS-CoV-2 infection?

  • Mass spec approach to study protein abundance and phosphorylation
  • Viral proteins increase after 8 hours, indicating viral replication
  • Small changes to host proteins within 24 hours
  • Large changes to the host phosphoproteome
• What are protein-protein interactions and their importance?

  • Many proteins function as part of large complexes
  • Important for understanding function and changes during disease
  • Blocking interactions may be a potential drug target
• What is required for the assembly of macromolecular complexes?

  Assembly requires two or more macromolecules, with at least one being a protein.
• What is the "bottom-up" approach in proteomics?

  The "bottom-up" approach involves enzymatic digestion of proteins prior to analysis by mass spectrometry.
• What are the challenges in characterizing large protein complexes?

  • Complexes are very large (MDa range)
  • Most size exclusion columns are ineffective
  • Complexes can be fragile and must be isolated under near-native conditions
• What is co-immunoprecipitation (Co-IP) used for?

  Co-IP is used to purify protein complexes by adding tags to proteins.
• What is the purpose of mass spectrometry in proteomics?

  Mass spectrometry is used to identify proteins and characterize their amino acid sequences.
• What are some tags used in Co-IP?

  Tags include TAP-tag, strep-tag, His-tag, and Flag-tag.
• What are the advantages of Co-IP by TAP-tagging?

  It enables purification under native conditions and provides higher purity through two affinity purification steps.
• How does a mass spectrometer separate ions?

  A mass spectrometer separates ions based on their mass to charge ratio (m/z).
• What are the disadvantages of Co-IP by TAP-tagging?

  It requires tags that can interfere with function and may not work well for low abundant complexes.
• How is complex identification achieved by mass-spectrometry?

  • Compare to non-tagged control
  • Identify proteins enriched in the sample
  • Verify interactions using other experiments
• What are the advantages of mass spectrometry as a technique?

  Mass spectrometry is fast, sensitive, and specific for determining molecular weight and mapping post-translational modifications.
• What is the role of protein degradation in bacteria?

  It involves the removal of damaged proteins and is a key regulatory feature.
• What is metabolomics?

  • Large-scale study of small molecules (metabolites)
  • Reflects biochemical activity and state of cells/tissues
  • Collectively known as the metabolome
• What is the ideal mass range for peptide fragments in mass spectrometry analysis?

  The ideal mass range for peptide fragments is between 700 to 2000 Da.
• How many metabolites are estimated to exist across the plant kingdom?

  It is estimated that there are over 1,000,000 metabolites across the plant kingdom.
• What does trypsin do in proteomics?

  Trypsin cleaves proteins at basic arginine (R) or lysine (K) amino acid residues.
• How many metabolites are found in the human metabolome database?

  There are 217,920 metabolites in the human metabolome database.
• What are the challenges associated with metabolomics?

  Challenges include chemical diversity, high turnover, and broad dynamic range.
• What is the purpose of quantitative proteomics?

  Quantitative proteomics aims to determine the relative and absolute amounts of proteins in samples.
• What are the pros and cons of targeted versus untargeted metabolomics?

  Targeted:

  • Pros: Absolute quantification, higher accuracy, pathway mapping
  • Cons: Not comprehensive, slower throughput

  
  

  Untargeted:

  • Pros: Comprehensive, biomarker discovery, higher-throughput
  • Cons: Semi-quantitative, many unknowns