Related Experiment Video
Updated: May 14, 2026
08:09
An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis
Published on: September 15, 2015
8.8K
Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics
Journal of the American Society for Mass Spectrometry
|July 10, 2024
Summary
Quenching protease activity after protein digestion is often unnecessary in proteomics. Using protein filter-trap methods allows direct peptide labeling, enhancing efficiency and automation for mass spectrometry analysis.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Standard proteomics workflows require quenching enzymatic digestions to prevent protease activity, which can cause overdigestion and interfere with isotopic quantitation.
- Postdigestion acidification, a common quenching method, is incompatible with chemical and isobaric labeling strategies (e.g., TMT/iTRAQ), necessitating additional steps like buffer exchange and pH adjustment.
- These extra steps reduce sample recovery, lower throughput, and complicate automation in mass spectrometry-based proteomics.
Purpose of the Study:
- To investigate whether quenching is necessary when generating peptides using protein filter-trap methods.
- To determine if direct peptide labeling is feasible after filter-trap digestion, bypassing traditional quenching steps.
- To assess the impact of eliminating quenching on sample recovery, processing time, and automation potential in proteomics.
Main Methods:
- Peptides were generated from proteins captured on filter traps.
- Trypsin activity in the eluate from filter-trap preparations was assessed.
- Direct labeling of peptides was performed without postdigestion quenching or pH adjustment.
Main Results:
- Residual trypsin activity and intact trypsin were found to be negligible in the eluate from protein filter-trap preparations.
- Direct labeling of peptides was successfully performed on enzymatic digests obtained via filter-trap methods.
- This streamlined approach improved sample recovery, increased throughput, and facilitated easier automation.
Conclusions:
- Quenching enzymatic digestions is unnecessary when using protein filter-trap methods in proteomics.
- Directly labeling peptides from filter-trap digests simplifies workflows and is compatible with chemical and isobaric labeling techniques.
- Eliminating the quenching step enhances the efficiency, recovery, and automation of mass spectrometry-based proteomics analyses.
More Related Videos
10:37Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
11.9K
11:12Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry
Published on: February 7, 2022
10.1K
Related Concept Videos
01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:16In vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:58Mechanical Protein Function
Proteins perform many mechanical functions in a cell. These proteins can be classified into two general categories- proteins that generate mechanical forces and proteins that are subjected to mechanical forces. Proteins providing mechanical support to the structure of the cell, such as keratin, are subjected to mechanical force, whereas proteins involved in cell movement and transport of molecules across cell membranes, such as an ion pump, are examples of generating mechanical force.
01:17The Proteasome Structure
The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an...
The proteasome is an...
01:21Overview of Protein Metabolism
Proteins are broken down into amino acids during digestion. Unlike fats and carbohydrates, which are stored for later use, proteins are not. Instead, amino acids are either used to produce ATP through oxidation or contribute to the creation of new proteins for the growth and repair of the body. Any surplus amino acids from the diet are converted into glucose or triglycerides rather than excreted.
Amino acids play various roles in the body once they are absorbed into cells. They are restructured...
Amino acids play various roles in the body once they are absorbed into cells. They are restructured...
01:18Amino Acid Catabolism
Microorganisms rely on proteins as an essential carbon and energy source, particularly in environments with limited polysaccharides or lipids. However, proteins are too large to cross the plasma membrane unaided, necessitating enzymatic degradation. Microbes secrete extracellular proteases and peptidases that hydrolyze proteins into peptides, which can then be transported across the membrane. Once inside the cell, intracellular proteases degrade these peptides into free amino acids, which...