Untitled Flashcards Set

Proteome

The overall protein content of a cell.

Proteomics

The characterization of the proteome, including expression, structure, interactions, and modifications.

Mass Spectrometry (MS)

A technique that reveals the quantitative state of a proteome and enables high throughput proteome analysis by breaking down proteins into smaller parts.

Trypsin

An enzyme used to break down positive amino acids in proteins during mass spectrometry.

iTRAQ

Isobaric tags for relative and absolute quantification used in mass spectrometry to analyze multiple samples simultaneously.

TMT

Tandem mass tags, a technique used in mass spectrometry for multiplexing samples.

Post-Translational Modifications (PTMs)

Chemical modifications of proteins after translation, affecting their function and activity.

Phosphorylation

The most common post-translational modification, involving the addition of a phosphate group to a protein.

examples of post translational modifications

include methylation, acetylation, and ubiquitination.

Co-Immunoprecipitation (co-IP)

A technique used to pull down a protein of interest along with its interacting partners using a specific antibody.

BioID

Proximity-dependent biotin identification method which uses a bacterial enzyme to tag interacting proteins with biotin.

Phage display

A technique to engineer phages to display proteins on their surface, allowing for the identification of protein interactions.

Yeast 2 hybrid

A method used to study protein-protein interactions by using a reporter gene activated by interacting proteins in yeast.

Protein-Protein Interaction Proteomics

A study of protein function by establishing relationships between proteins.

methods to study protein protein interactions

include techniques like phage display and yeast two-hybrid.

Co-Immunoprecipitation

Protein complementation assay

A method that uses two different proteins with observable activity to indicate interaction by producing a visible signal.

The Human Protein Atlas

A project that maps all proteins in the human body, detailing their expression and tissue localization.

Glycosylation

A post-translational modification involving the addition of sugar moieties to proteins, affecting their stability and function.

Lectins

Proteins that specifically bind sugars, used in the identification of glycosylated proteins.

Acetylation

A post-translational modification that involves the addition of an acetyl group, influencing protein stability and interactions.

Anti-Acetyl-Lysine Antibodies

Antibodies that specifically bind to acetylated lysine residues, used for the detection of acetylation.

Metal-Based Affinity Purification

A method to isolate phosphorylated proteins by taking advantage of their negative charge due to phosphate groups.

Comparison of Phosphorylated and Unphosphorylated Proteins

A technique to identify common post-translational modifications by examining the protein differences caused by phosphorylation.

Understanding Protein Activities through PTMs

Knowledge of post-translational modifications enhances the understanding of protein function and regulation.

Identifying Protein Modifications

Using techniques like mass spectrometry to determine the type and quantity of modifications present on proteins.

Cross-Linking

A method used to covalently bond proteins together to stabilize interactions for analysis, although it may generate false positives by linking non-interacting proteins.

Proximity-Dependent Biotin Identification (BioID)

A technique that uses the bacterial enzyme BirA to biotinylate proteins in proximity to a target protein, allowing for the identification of interactors.

biotin-avidin interaction

One of the strongest connections in nature, where biotin binds strongly to avidin, used in BioID to isolate tagged proteins.

Phage Display

A method that uses engineered phages to display target proteins on their surface, allowing for the identification of protein interactions.

High Throughput Screening

A process that allows the rapid testing of many samples, exemplified in phage display methods for protein interaction analysis.

Protein Tagging

The addition of a specific sequence to a protein to facilitate purification, identification, or quantification, commonly used in various proteomics techniques.

Tagging Proteins with BirA

Fusing BirA to a protein of interest allows for the biotinylation of interacting proteins, aiding in their identification.

Western Blot (WB)

A technique used to detect specific proteins in a sample, often following immunoprecipitation methods like co-IP.

Mass Spectrometry (MS) Analysis

A technique used to analyze the mass and composition of proteins, often employed after immunoprecipitation or tagging methods.

Protein Interactions Mapping

A process aimed at identifying and detailing interactions between proteins in a cell, often involving techniques like BioID or phage display.

Yeast Two-Hybrid System

A method used to study protein-protein interactions by using a reporter gene activated through the interaction of target proteins with fused transcription factors.

Transcription Factors

Proteins that bind to DNA and activate the transcription of specific genes, requiring both a DNA-binding domain and an activation domain.

DNA-Binding Domain

The part of a transcription factor that binds to specific DNA sequences associated with the reporter gene.

Activation Domain

The portion of a transcription factor that helps recruit the transcriptional machinery, facilitating gene expression.

Open Reading Frame (ORF)

A sequence of DNA that can be translated into a protein; used here for expressing human proteins in yeast during the interaction study.

Artificial Interaction

A forced interaction between proteins in a laboratory setting that may not reflect their natural behavior in vivo.

Genome Sequencing in Yeast

The process of determining the sequence of proteins expressed in yeast to identify which proteins interacted with the target protein.

Intracellular Localization

The specific location of proteins within a cell, which is crucial for assessing the validity of interactions observed in experimental models.

Limitations of Yeast Two-Hybrid

The method's drawback of possibly forcing interactions that would not naturally occur in their native cellular environments.

Phage Display Limitations

The concern that in phage display, protein interactions might be artificially induced and not representative of true biological interactions.

Protein-Protein Interaction Proteomics (Protein Complementation Assay)

A method that uses two different proteins with observable activity (such as fluorescence or antibiotic resistance) that only creates this activity when the proteins interact. It is performed in human cells and is less artificial than other methods like yeast 2 hybrid.

Protein Tagging in Protein Complementation Assay

In this method, the target protein A is tagged, while different protein B is represented in each cell from the human open reading frame. Only interacting proteins result in observable activity.

Sequencing of Glowing Cells in Protein Complementation Assay

Cells that exhibit fluorescence (activation due to interaction with the target protein) can be sequenced to identify which protein is expressed.