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Intact protein barcoding enables one-shot identification of CRISPRi strains and their metabolic state

ABSTRACT: Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow-injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed 6 ubiquitin-barcoded CRISPRi strains targeting metabolic enzymes, and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.

INSTRUMENT(S): 6546 Q-TOF LC/MS (Agilent instrument model)

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Prof. Hannes Link

PROVIDER: MSV000096254 | MassIVE | Wed Oct 30 13:51:00 GMT 2024

REPOSITORIES: MassIVE

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