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Protocol for absolute quantification of proteins in Gram-negative bacteria based on QconCAT-based labeled peptides
Nicolás Gurdo1, Shannara Kayleigh Taylor Parkins1, Martina Fricano1
1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
STAR Protocols
|February 28, 2023
Summary
This study presents a method for protein quantification using labeled quantification concatamer (QconCAT) as internal standards. The workflow enables accurate measurement of soluble proteins in Pseudomonas putida KT2440.
Area of Science:
- Proteomics
- Analytical Chemistry
- Molecular Biology
Background:
- Mass spectrometry-based absolute protein quantification relies on internal standards (ISs).
- Quantification involves comparing ion intensity ratios between analytes and cognate ISs.
- Labeled quantification concatamer (QconCAT) serves as a common IS for this technique.
Purpose of the Study:
- To describe a systematic workflow for designing, producing, and purifying QconCATs.
- To apply this methodology for quantifying soluble proteins in Pseudomonas putida KT2440.
- To establish a versatile platform for producing ISs applicable to diverse biological systems.
Main Methods:
- Design, synthesis, and purification of QconCATs.
- Mass spectrometry-based analysis of protein samples.
- Calculation of absolute protein amounts using QconCATs as internal standards.
Main Results:
- A systematic protocol for QconCAT generation and purification was established.
- Soluble proteins in Pseudomonas putida KT2440 were successfully quantified.
- The methodology demonstrated the quantification of detectable peptides.
Conclusions:
- The developed workflow provides a robust method for QconCAT production.
- This approach enables accurate absolute protein quantification in complex biological samples.
- The platform is adaptable for generating internal standards for various proteomic studies.
