PROTEOMICS KEY POINTS

What is proteomics?

The study of large scale proteins, using high throughput technology

What type of approach does Proteomics employ to capture different aspects of protein biology?

Complementary proteomic approaches

How is high sensitivity achieved in antibody based proteomics?

Through signal amplification

What ensures the specificity of antibody based proteomics?

Validated antibodies

What ensures the reproducibility of Antibody-Based Proteomics assays?

All samples printed on the same slide; rigorous assay controls.

What types of proteins are often low-abundance and require highly sensitive technologies for detection?

Regulatory and post-translationally modified proteins.

What molecular interaction forms the basis of detection in Antibody-Based Proteomics?

Antigen-antibody interactions.

What is the role of fluorescent dyes in the detection process?

To visualize and enhance sensitivity.

Name two examples of catalyzed signal amplification used to increase detection.

TSA (HRP enzyme) or biotin-streptavidin binding.

What is the typical high-throughput capacity of an RPPA slide (in samples and total spots)?

~ 3000

What is the defining specificity feature of Reverse Phase Protein Array (RPPA) regarding antibodies per slide?

One antibody is used per entire slide (∼300 total antibodies are used across different slides for an assay).

RPPA is usually used for which types of samples?

Tissue or cell line samples.

After protein extraction for RPPA, what is the next step before detection?

The protein is spotted on glass slides

Luminex is usually used for which types of samples?

Serum or plasma samples

In Luminex, how are target proteins bound? (Priniciple)

Antibodies are conjugated to beads, which then bind the target proteins

How is detection achieved in Luminex?

Through fluorescent dye and signal amplification

What factor often determines the choice between RPPA and Luminex?

The availability of validated antibodies

What is the overall purpose of positive and negative controls in the experiments?

Controls confirm antibody specificity and validate assay reliability

What is the positive control in antibody based proteomics?

Making sure the primary antibody is only binding to protein of interest

What is the negative control in antibody based proteomics?

Instead of a primary antibody, use a buffer to see if the secondary binds to anything. If you see a band you subtract the flurorscent intensity from the actual treatment

Which technology is most appropriate for identifying protein differences between normal and cancerous tissue samples (e.g., lung cancer)?

Reverse Phase Protein Array (RPPA) 

What is the detection mechanism for RPPA after the antigen-antibody interaction?

Fluorescent dye and signal amplification

What is extracted from tissue samples before being spotted onto an RPPA slide for analysis?

Proteins 

RRPA is ____

intracellular

Luminex is ____

extracellular

The principle of RPPA 

Protein is extracted, spotted on glass slides, and detected using antibody–antigen interactions with fluorescent dye and signal amplification

Example #1: A surgeon has operated on 20 lung cancer patients, preserving a piece of lung cancer tissue in one tube and an adjacent piece of normal lung tissue in another. The objective is to identify protein and phosphorylated protein changes between normal and cancerous lung tissues to determine common alterations. Protein will be extracted from each tissue sample, yielding 20 samples from normal lung tissue and 20 from cancerous lung tissue.

Which technology would be used?

RPPA

For 300+ proteins or phosphorylated proteins will then be employed to identify proteins that differ between the two groups

Example #2: A research lab is studying COVID-19 and aims to determine which immune markers are altered in patient blood. Serum samples were collected directly from 20 COVID-19 positive patients and 20 healthy volunteers, resulting in 40 total samples.

Which technology would be used?

Luminex

Researchers will assay 80 cytokines and other immune markers from these serum samples in a single experiment. The objective is to identify cytokines and chemokines that consistently differ between COVID-19 patients and healthy controls, uncovering immune signatures and potential biomarkers linked to disease severity and progression.