Proteolysis Analysis

An important method of proteolysis is the breakdown of proteins into individual amino acids. Proteolysis analysis services are provided by Creative Proteomics to help clients better understand the mechanisms and purposes of proteolysis, which is an important addition to the research of related subjects.

What is Proteolysis?

In life on earth, proteins are broken down into peptides and amino acids by a process called proteolysis. It is a protracted process that is accelerated by particular proteases, which are found in both bacteria and plants but are most prevalent in animals. Protein bond cleavage could take hundreds of years without these enzymes.

Fig 1. Protein–small molecule interactions control many cellular processes, such as allosteric regulation of enzyme activity. (Pepelnjak, M., et al.; 2020)

It is an essential activity that supports cellular activities like metabolic regulation, cell structure maintenance, and transportation in both humans and animals. Another important factor in the development of various disorders is proteolysis. It also has a few industrial uses, including the processing of food and stain treatment.

Challenges in Proteolysis Analysis

1. Sample Complexity: Proteomes exhibit extensive complexity, encompassing an array of thousands of distinct proteins manifesting across divergent abundance levels. The entailing complexity trial makes it arduous to accurately detect and scrutinize proteolytic events.
2. Temporal Dynamics: Proteolysis represents a dynamic process that can ensue transiently and rapidly in reaction to cellular signals or provocations. Such transient nature necessitates fine-tuned timing and rigorous sample collection methodologies.
3. Detection Methods: Conventional protein analysis methodologies, such as gel-based electrophoresis and mass spectrometry, may not consistently deliver adequate sensitivity or resolution for identification and quantification of proteolytic fragments. Thus, it is imperative to develop more sensitive, specific detection methods.
4. Protease Specificity: Intracellular environment is populated by a rich diversity of proteases, each having its unique substrate specificity. Comprehending the specific proteases involved in a given proteolytic event along with their target substrates is a critical factor for credible proteolysis study. This initiates the challenge of creating new tools and techniques for the characterization of protease-substrate interactions.
5. Data Analysis and Interpretation: Proteolysis analysis yields an overwhelming volume of proteomics data, consolidating peptide quantification and identification. This necessitates the use of advanced bioinformatics tools and strategies. Hence, data integration and proper interpretation serve as continuous challenges in understanding the intricate regulatory networks and resultant functional repercussions of proteolysis.

Overcoming these challenges in proteolysis analysis is crucial for advancing our understanding of protein regulation and function, as well as for identifying potential therapeutic targets in various diseases.

What can we offer?

Gel-based techniques: Gel electrophoresis techniques, such as SDS-PAGE or native gel electrophoresis, can be used to separate proteins based on their size. Changes in protein band patterns or appearance of new bands can indicate proteolysis.

Mass spectrometry (MS): MS can identify and quantify proteolytic fragments by analyzing the mass-to-charge ratio of peptide ions. Techniques like liquid chromatography-tandem mass spectrometry (LC-MS/MS) are commonly used for proteolysis analysis.

Activity-based probes: These are small molecule probes that can selectively label active proteases. By using fluorescent or biotin tags, protease activity can be detected and visualized.

Western blotting: Antibodies specific to intact proteins or proteolytic fragments can be used to detect their presence in a sample. Changes in band intensity or size can indicate proteolysis.

Fluorescence resonance energy transfer (FRET) assays: FRET-based assays utilize fluorescence signals emitted when two fluorophores are in close proximity. By incorporating specific protease recognition sequences (substrates), proteolytic activity can be measured.

Service Workflow

Our Commitment and Future Goals

Creative Proteomics is committed to helping our clients meet the growing and changing demands for protein drug characterization. We offer a wide range of services related to protein drug characterization and ensure quality and reliability of results as well as on-time delivery. If you are interested in our services or have any additional questions, please feel free to contact us, we are pleased to hear from you and look forward to working with you.

Reference

1. Pepelnjak, M., et al.; Detecting Protein–Small Molecule Interactions Using Limited Proteolysis–Mass Spectrometry (LiP-MS). Trends in Biochemical Sciences. 2020.

For research use only, not intended for any clinical use.