Comparative Proteomics Lecture Notes

These flashcards cover key terms and definitions from the Comparative Proteomics lecture, helping students grasp fundamental concepts and terminology in proteomics.

Proteomics

The study of proteins, particularly their structures and functions.

What is the primary focus of proteomics research beyond just identifying proteins?

Understanding the functions, structures, interactions, and modifications of proteins, especially their roles in diseases.

What are the main challenges in studying the proteome?

The high dynamic range of protein concentrations, diversity from posttranslational modifications and alternative splicing, and rapid changes over time.

Proteome

The entirety of proteins in existence in an organism throughout its life cycle, or in a particular cell type.

How does the proteome differ from the genome?

The genome is relatively static, representing all genes. The proteome is dynamic, reflecting the subset of genes being expressed and modified in a specific cell at a given time.

What factors contribute to the complexity and variability of the proteome?

Alternative splicing, posttranslational modifications, RNA editing, and varying protein expression levels due to cellular conditions or developmental stages.

Why is quantifying proteins important in proteomics?

To determine the abundance of proteins, which can indicate their functional significance and changes in disease states or drug responses.

Alternative Splicing

The process by which different combinations of exons are included in the final mRNA transcript, leading to multiple protein variants from a single gene.

How does alternative splicing contribute to protein diversity from a single gene?

By selectively including or excluding certain exons from the primary RNA transcript, leading to different mRNA variants and subsequently different protein isoforms.

Posttranslational Modification

Chemical modifications occurring to proteins after translation, affecting their functions.

What is an example of a common posttranslational modification?

Phosphorylation, where a phosphate group is added to a protein, often altering its activity or interactions.

What is the role of phosphorylation in cellular signaling?

It often acts as a molecular switch, turning protein activity on or off, thereby regulating signal transduction pathways.

What are other significant types of posttranslational modifications?

Glycosylation (addition of sugars), ubiquitination (targeting for degradation), and acetylation (modifying histone proteins).

SDS-PAGE

A method to separate proteins based on their size using polyacrylamide gel electrophoresis with sodium dodecyl sulfate.

What is the primary principle behind protein separation in SDS-PAGE?

Proteins migrate through a polymer gel based primarily on their molecular weight after being denatured and coated with a uniform negative charge by SDS.

What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE?

SDS is an anionic detergent that denatures proteins and confers a uniform negative charge-to-mass ratio, allowing separation based solely on size.

Why are proteins typically denatured before SDS-PAGE?

To unfold them into linear chains, ensuring that their migration through the gel is independent of their native shape or intrinsic charge.

What is the role of a reducing agent (e.g., \beta -mercaptoethanol or DTT) in SDS-PAGE sample preparation?

It breaks disulfide bonds within or between protein subunits, ensuring complete denaturation and separation based on individual polypeptide chain size.

Why is a polyacrylamide gel used in SDS-PAGE?

The polyacrylamide gel acts as a molecular sieve, allowing proteins to be separated based on their size as they migrate under an electric field through pores of a specific size.

Western Blotting

A technique used to identify specific proteins in a sample using gel electrophoresis followed by antibody binding.

What are the main steps involved in a Western Blotting procedure?

SDS-PAGE separation, transfer of proteins to a membrane, blocking of non-specific binding sites, incubation with primary antibody, incubation with secondary antibody, and detection.

What is the role of the primary antibody in Western Blotting?

It specifically binds to the target protein of interest on the membrane.

What is the role of the secondary antibody in Western Blotting?

It binds to the primary antibody and is conjugated to an enzyme or fluorophore that allows for the detection of the protein-antibody complex.

What is the purpose of blocking agents (e.g., milk or BSA) in Western Blotting?

They cover unoccupied sites on the membrane, preventing non-specific binding of the primary or secondary antibodies and reducing background signal.

How are proteins typically transferred from the gel to a membrane in Western Blotting?

Using an electric current to move the separated proteins from the polyacrylamide gel onto a PVDF or nitrocellulose membrane, where they become immobilized.

What common types of membranes are used for Western Blotting protein transfer?

Polyvinylidene fluoride (PVDF) or nitrocellulose membranes.

What is the function of Tris-buffered saline (TBS) or Phosphate-buffered saline (PBS) in Western blotting?

These buffers are used for diluting antibodies and washing membranes to maintain physiological pH and reduce non-specific binding.

What is Horseradish Peroxidase (HRP) commonly used for in Western Blotting?

HRP is an enzyme conjugated to secondary antibodies, which catalyzes a chemiluminescent reaction for signal detection when a specific substrate is added.

What are common challenges or sources of background in Western Blotting?

Non-specific antibody binding, insufficient washing, and high antibody concentrations, which can obscure target protein detection.

What are some common detection methods used after secondary antibody binding in Western Blotting?

Chemiluminescence (using an enzyme like HRP to produce light), fluorescence (using a fluorophore), or chromogenic detection (producing a colored precipitate).

What are some common applications of Western Blotting?

Quantifying protein levels, detecting posttranslational modifications, confirming protein expression, and identifying protein-protein interactions.

Myosin Light Chain

A type of protein involved in muscle contraction, with variations existing among different species.

Why is the Myosin Light Chain a good example for comparative proteomics or evolutionary studies?

Its variations across species can provide insights into evolutionary relationships and adaptations in muscle function.

What was the specific objective involving Myosin Light Chain in this lab context?

To compare Myosin Light Chain 2 (MLC2) expression or variation across different fish species using Western blot analysis to infer evolutionary relationships or functional adaptations.

Cladogram

A branching diagram that illustrates the evolutionary relationships among organisms based on shared characteristics.

What types of data are typically used to construct a cladogram?

Morphological features, genetic sequences (DNA or protein), biochemical characteristics, and fossil evidence.