Proteomics Overview and Methods

Proteomics Overview

• Proteomics is the study of proteins and their interactions, function, and structures within cells.
• It provides a more comprehensive understanding of biological systems compared to genomics alone.
• Proteomics helps characterize proteins, understand protein interactions, and identify disease biomarkers.
• Proteomics is a rapidly developing field, especially in therapeutics.
• "Proteome" was coined by Marc Wilkins in 1995.

Lecture Outlines

• Analysis of protein sequences and structures.
• Proteomics methods.
• 2D gel electrophoresis - a key method in proteomics.
• Comparing RNA and proteomic data.

Protein Structure Levels

• Primary structure: determined by the sequence of amino acids.
• Secondary structure: formed by hydrogen bonds between amino acids.
• Tertiary structure: the overall three-dimensional shape of a protein.
• Quaternary structure: complex formed by more than one polypeptide chain.

Proteomics Meaning

• Combining protein and genomics data to improve our understanding.
• Proteomics aims to understand the structure, function, and interactions of proteins.

Proteomics Applications

• Expression Profiling: Analyzing protein expression levels in different conditions (e.g., disease vs. healthy states).
• Disease Diagnosis: Using specific protein signatures to identify diseases.
• Biomarker Detection: Identifying molecules that may indicate disease.
• Protein Complexes: Investigating interactions and assemblies between proteins.
• Drug Discovery: Identifying proteins as targets for drug development.
• Protein Function: Determining the role and activity of different proteins.
• Protein Interactions: Studying how proteins interact and work together.
• Protein Engineering: Modifying proteins or designing new ones for specific purposes.
• Post-translational Modifications: Studying changes to proteins after synthesis.
• Functional Proteomics: Studies protein functions and the molecular mechanisms in cells.
• Structural Proteomics: Understanding three-dimensional shape of proteins.

Types of Proteomics

• Expression proteomics: Studying the quantity and type of proteins under various conditions.
• Structural proteomics: Analyzing the 3D structure of proteins.
• Functional proteomics: Determining the role and function of proteins.

Importance of Proteomics

• Proteins, not genes, drive cellular phenotypes.
• Proteomics is essential for understanding disease mechanisms, aging, and environmental impacts.
• Information from studying proteins alone complements information gained from studying genes.

Proteomics Tools

• Conventional Techniques: Chromatography, western blotting, size exclusion, etc.
• In-silico Techniques: Computational approaches (e.g., sequence alignment)
• Advanced Techniques: Mass spectrometry (e.g., tandem MS, DIGE), protein microarrays.
• Quantitative Techniques: ICAT, SILAC, iTRAQ.
• Protein Analysis Techniques: Edman sequencing, X-ray crystallography, NMR spectroscopy.
• Purification Techniques: Chromatography-based techniques.
• Analysis Techniques: ELISA, Western blotting, protein microarrays.
• Characterization Techniques: Gel-based approaches, mass spectrometry.
• Quantification Techniques: ICAT, SILAC, iTRAQ.
• Sequence Analysis: Edman sequencing.

Protein Sequence and Structure Analysis

• Protein sequence analysis: a method for studying protein characteristics.
• Techniques include sequence alignments with other proteins and comparing against biological databases.
• Studying protein sequence helps to assign functions through similarities.
• High-throughput technologies accelerate the creation of databases, which aid in better understanding organismal biology.

Tandem Mass Spectrometry (MS)

• A commonly used high-throughput proteomics technology.
• Tandem MS involves multiple mass analyzers to identify fragments, aiding protein sequencing.
• Techniques distinguish different peptide types.
• Data analysis tools facilitate the identification of known proteins or the discovery of new ones.

2D Gel Electrophoresis (2DE)

• A method for separating proteins based on charge (pI) and size.
• Widely used for protein expression profiling in proteomic experiments.
• 2DE can identify changes in protein expression due to disease or drug treatment and post-translational modifications.

In silico Approach

• Utilizing computational tools for protein research.
• Resources like EMBL-EBI databases (e.g., UniProtKB, IntAct, AlphaFold, PRIDE) house crucial protein data.
• UniProtKB: A cornerstone protein database, with categorized data types for different analyses.

AlphaFold Protein Structure Database

• Artificial intelligence-based program developed by DeepMind to predict protein structures.
• AlphaFold 3 utilizes a sophisticated deep learning architecture to make accurate predictions.
• The database is accessible for non-commercial research to accelerate scientific discovery.

Comparisons between RNA and Proteomic Data

• RNA-Seq derived protein databases better approximate actual cellular/tissue protein pools, thereby enhancing protein identification accuracies.
• Proteomic data confirms the validity and relevance of findings from RNA-seq data.

Traditional and RNA-Seq-based Proteogenomics

• Traditional methods rely on predicted protein sequences from genomic data using six-frame translation and gene models.
• RNA-Seq methods use RNA sequencing to confirm or re-assess predicted coding sequences, identify novel splice variants, or discover novel genes that would be missed in traditional methods.

Explore the essential concepts of proteomics, including protein structures, interactions, and various methods utilized in analyzing them. This quiz will deepen your understanding of how proteomics enhances our knowledge of biological systems and disease biomarkers. Discover how techniques like 2D gel electrophoresis play a vital role in this rapidly evolving field.