Protocol for absolute quantification of proteins in Gram-negative bacteria based on QconCAT-based labeled peptides

Nicolás Gurdo; Shannara Kayleigh Taylor Parkins; Martina Fricano; Tune Wulff; Lars Keld Nielsen; Pablo Iván Nikel

doi:10.1016/j.xpro.2023.102060

Protocol for absolute quantification of proteins in Gram-negative bacteria based on QconCAT-based labeled peptides

STAR Protoc. 2023 Mar 17;4(1):102060. doi: 10.1016/j.xpro.2023.102060. Epub 2023 Jan 24.

Authors

Nicolás Gurdo¹, Shannara Kayleigh Taylor Parkins¹, Martina Fricano¹, Tune Wulff¹, Lars Keld Nielsen², Pablo Iván Nikel³

Affiliations

¹ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
² The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
³ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark. Electronic address: [email protected].

Abstract

Mass-spectrometry-based absolute protein quantification uses labeled quantification concatamer (QconCAT) as internal standards (ISs). To calculate the amount of protein(s), the ion intensity ratio between the analyte and its cognate IS is compared in each biological sample. The present protocol describes a systematic workflow to design, produce, and purify QconCATs and to quantify soluble proteins in Pseudomonas putida KT2440. Our methodology enables the quantification of detectable peptide and serves as a versatile platform to produce ISs for different biological systems.

Keywords: Biotechnology and Bioengineering; Mass Spectrometry; Microbiology; Protein Biochemistry; Protein Expression and Purification; Systems Biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gram-Negative Bacteria / metabolism
Mass Spectrometry
Peptides* / metabolism
Proteins
Proteomics* / methods

Substances

Peptides
Proteins