proteomics & bioinformatic tools for protein studies

what is bioinformatics?

• scientific subdiscipline that involves using computer technology to collect, store, analyze, and disseminate biological data and information, such as DNA and amino acid sequences or annotations about those sequences

• scientists and clinicians use databases that organize and index such biological information to increase our understanding of health and disease, and in certain cases, as part of medical care

• interdisciplinary field

what are the applications fo bioinformatics?

• identifying new drug targets
• understanding disease mechanism
• designing new drugs
• predicting interactions between a compound and an enzyme
• predicting drug response/safety
• streamlining clinical trials
• shortening timeline and reducing the cost of drug development
• reducing the risk of side effects
• fostering the growth of personalized medicine

what is the relevance of bioinformatics to pharmacology?

• use of genomics, genetics, proteomics, and transcriptomics in target discovery
• use of chemogenomics, structural genomics, and homology modeling in target and drug discovery
• use of biomarkers, toxicogenomics, pharmacogenomics, in silico modeling in drug development

what is kerendia (finerenone)?

• selective, nonsteroidal mineralocorticoid receptor antagonist
• bayer
• example of developing a therapeutic hypothesis based on genetic evidence
• indicated for CKD and type 2 diabetes
• also indicated for microalbuminuria: increased levels of the urinary albumin-to-creatinine ratio (UACR); early indication of CKD

what is the MOA of kerendia (finerenone)?

• NR3C2: gene encodes the mineralocorticoid receptor associated with microalbuminuria
• blocks the MR
• reduces the risk of CKD and slows it down from getting worse

what are the bioinformatics techniques?

• sequence analysis
• structural bioinformatics
• high-throughput techniques
• biomedical image-based analysis
• network and systems biology

what are the bioinformatics tools?

• databases: sequence storage databases
• computational models: mathematical models to describe biological systems
• software tools: sequence alignment, function analysis, and clinical tools; image analysis software
• machine learning applications: protein structure and drug response predictions

what is UniProt?

• freely accessible database of protein sequences and functional information
• hub for protein information

what is the information provided by UniProt?

• general information of a protein of interest
• subcellular location
• diseases caused by variants/mutations in the gene
• pharmaceutical: use of a protein as a pharmaceutical drug
• post-translational modifications/processing
• protein structure

what is the importance of determining the protein structure?

• medicine: drug design, predict the function of a mutated protein
• biochem: predict the mechanism of action and develop tools to manipulate it
• biotech: design of new enzymes

what are the techniques used to determine the protein conformation?

• X-ray crystallography: uses diffracted X-rays from a protein crystal to generate an electron density map, which indicates the atomic positions of proteins that can readily crystallize
• nuclear magnetic resonance (NMR) spectroscopy: reveals the structure and dynamics of proteins in solution by identifying protons in close proximity to one another
• cryo-electron microscopy: a rapidly developing method that can elucidate the structures of lare multimeric complexes at increasingly higher resolutions

what are the approaches to predict 3D structures?

• ab initia predictions: without prior knowledge; calculations that attempt to minimize the free energy of a structure
• knowledge-based methods: an unknown primary structure is examined for compatibility with known protein structures/fragments

what is protein structure prediction?

• one of the most important goals of computational biology
• extremely challenging

what are the computational structural prediction methods of 3D structures of biomolecules?

• homology modeling: to predict the structure of an unknown protein from existing homologous proteins
• protein-ligand docking and virtual screening: to predict and analyze the binding interactions between small molecules (ligands) and proteins

what is relenza (zanamivir)?

• treatment of illness due to influenza A and B virus in adults and pediatri patients aed 7 yrs of age and older who have been symptomatic for no more than 2 days
• structure-based design: selecting molecules that were likely to bind to the conserved regions of the enzyme neuraminidase

what is neuraminidase?

enzyme produced by the flu virus to release newly formed virus from infected cells

what is AlphaFold?

• significant step forward in protein folding prediction
• structures are vastly more accurate than competing methods
• freely available, AI program that can now predict the shape of a protein, almost instantly, down to atomic accuracy
• predictions are valuable hypotheses and accelerate but do not replace experimental structure determination

what is DeepMind?

software that can predict the 3D shape of proteins

what are the limitations of AlphaFold?

• cannot predict the consequences of new mutations in proteins, since there are no evolutionarily related sequences to examine
• cannot deal with proteins that can adopt different structures in different states/environments
• cannot predict protein structures bound to ligands

what is Isomorphic Labs?

• DeepMind's drug-discovery spin-off
• predict the structures of proteins when they are bound to drugs and other interacting molecules

what is the Protein Data Bank (PDB)?

• RCSB PDB
• structures are published and can be accessed for visualization and analysis

what is proteomics?

• large-scale study of proteins: investigation of the proteome
• explore the complete catalog of proteins expressed in a cell type at a given time point
• investigate how this inventory changes when the conditions are altered
• results: abundance of peptides, peptide modifications, and interactions between peptides
• technology: mass spectrometry, western blotting, and ELISA

what is a proteome?

• whole set of proteins expressed in a cell at a particular time
• not a fixed characteristic of the cell

what is not a good predictor of protein abundance?

transcriptomic data: a transcribed gene may be differentially translated/not translated, and different proteins have different degradation rate

what are protein methods?

• protein purification
• polyacrylamide gel electrophoresis and western blotting
• enzyme-linked immunosorbent assay (ELISA)

what are the limits of protein methods?

• limited in number of proteins studied per condition/assay
• limited to the detection of proteins for which an antibody is available

what is mass spectrometry-based proteomics?

used to investigate:

• when and where proteins are expressed
• rates of protein production, degradation, and steady-state abundance
• how proteins are modified
• the movement of proteins between subcellular compartments
• how proteins interact with one another

what is mass spectrometry?

• powerful technique for the identification of peptides and proteins
• allows the highly precise and sensitive detection of the mass of an analyte
• can detect molecular masses with a high degree of sensitivity and accuracy

what are mass spectrometers?

• convert the analyte into gas-phase ions
• applies electrostatic potentials to measure the m/z ratio
• consists of 3 components: ion source, mass analyzer, detector
• electrospray ionization (ESI): technique to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol

what is matrix-assisted laser desorption/ionization (MALDI)?

• technique to produce ions using a laser energy-absorbing matrix
• often combined with a time-of-flight detector

how can proteins be analyzed?

• can be specifically cleaved into small peptides to facilitate analysis
• sequencing of long peptides by mass spectrometry yields complex spectra that are difficult to interpret

how is an entire protein be sequenced?

• chemically/enzymatically cleaved to yield peptides
• peptides are ionized and have m/z ratio
• the fragment ion spectra are then assigned peptide sequences based on database comparison and protein sequences are predicted

what is the process of generic MS-based proteomics experiment?

1. sample fractionation

• SDS-PAGE → excised proteins

1. trypsin digestion

• peptide mixture

1. peptide chromatography and ESI
2. MS
3. MS/MS

what is the process of co-immunoprecipitation followed by MS to identify protein interactors?

1. cell lysates: total protein

• add Ab and incubate

1. Ag-Ab complex

• add protein A/G beads and incubate

1. Ag-Ab-bead complex (with other proteins)

• was abd elute

1. Ag-Ab-bead complex (no other proteins)

• purify

1. digestion
2. MS

what is saphnelo (annifrolumab)?

• astrazeneca
• example of developing a therapeutic heypothesis based on protein interactions
• monoclonal Ab used for the treatment of systemic lupus erythematosus (SLE)
• binds the type I interferon receptor (IFNAR1)
• blocks the activity of type I interferon
• no direct genetic evidence linking SLE and IFNAR1
• missence variants in TYK2, have been associated with SLE: kinase that physically interacts with IFNAR1

what is quantitative proteomics?

the measurement of the abundance of proteins across multiple conditions (healthy vs disease, untreated vs drug treated)

what are the applications of proteomics-based technologies?

• detections of various diagnostic markers
• candidates for vaccine production
• understanding pathogenicity mechanisms
• alteration of expression patterns in response to different signals/medications
• interpretation of fucntional protein pathways in different disease

what is a proteome-wide atlas of drug MOA?

• high-throughput quantitative proteomics
• 875 small-molecule compounds
• comprehensive profiling method to characterize the MOA of small molecules
• each compound altered the expression of 15 proteins
• revealed potential new targets for commonly used small molecules
• elucidated compound MOA and drug repurposing
• revealed off-target effects, which can increase efficiency and safety profiling in drug discovery

what is genomics?

• large scale study of DNA
• the study of all of a person's genes
• genome: an organism's complete set of DNA
• sequencing: determining the exact order of the bases in a strand of DNA

what is the human genome project?

• the sequence of the human genome has been completed
• started in 1990 and completed in 2003
• led at the NIH by the National Human Genome Research Institute
• comprised of approximately: 3 billion bp of DNA distributed among 24 chromosomes; 23,000 genes

what is sanger/dideoxy sequencing?

• a method using DNA polymerase, along with special chain-terminating nucleotides, called dideoxyribonucleoside triphosphates
• when incorporated into a growing DNA strand, they block further elongation of that strand

what is automated sanger sequencing of DNA?

• 4 diferent chain-terminating nucleotides have been chemically tagged with a different colored fluorescent label
• loaded onto thin capillary gels, which separate the reaction products into a series of distinct bands
• a detector records the color of each band
• a computer translates the information into a nucleotide sequence
• sequence of overlapping segments
• longer sequences are assembled from shorter pieces

what is next-generation sequencing (NGS)?

• allows sequencing of thousands to millions of DNA molecules simultaneously
• high speed, reduced cost
• a genome/other large DNA sample is broken into millions of short fragments
• the sequences are then amplified on a solid surface with covalently attached linkers

what is RNA sequencing/transcriptomics?

method to detect the presence and quantification of all the RNA molecules in a cell under specific conditions

what is the process of RNA sequencing/transcriptomics?

1. isolate RNA from cells or tissue of interest
2. select for RNA by filtering for sequences containing poly(A) tails
3. synthesize cDNA using reverse transcriptase
4. sequence cDNA molecules using an NGS method
5. use computational algorithms to assemble the sequencing data

what is a heatmap?

a graphical representation of data where values are depicted by color

what is clustering?

• task of classifying N objects (proteins) into k groups (clusters) in such a way that the objects within a group are similar to each other, but the groups are different from each other
• methods identify similar and distinct expression patterns

what is principal component analysis (PCA)?

• method for combining the properties of an object
• can simplify the data to minimize its effect
• reduces the data to only 2 or 3 principal components

what is a volcano plot?

• type of scatter plot
• useful for identifying events that differ significantly between 2 groups of experimental subjects
• it plots significance vs fold change