The wild-type and cra deletion mutant harboring the IPTG-inducible plasmid
pPtac-cra were grown in M9 medium plus glucose (5 g l−1) or M9 plus
acetate (0.75 g l−1) plus 0 or 10 μM IPTG. Harvested cells were washed and
lysed, and the extracted proteins were digested with trypsin as described previously (Malmstrom
et al, 2009 (link)). Then, 10 pmol of heavy labeled
reference peptide was added to the digests, each containing 100 μg of total peptide. After
desalting the peptides with macro-spin columns (Harvard Apparatus), an aliquot containing 1
μg of peptide was subjected to targeted mass spectrometry using previously specified
instrument settings (Picotti et al, 2009 (link)).
The total number of cells in the samples was determined by flow cytometry. Averages and standard
deviations were derived from glucose experiments for the wild-type cells and from glucose and
acetate experiments for other strains.
pPtac-cra were grown in M9 medium plus glucose (5 g l−1) or M9 plus
acetate (0.75 g l−1) plus 0 or 10 μM IPTG. Harvested cells were washed and
lysed, and the extracted proteins were digested with trypsin as described previously (Malmstrom
et al, 2009 (link)). Then, 10 pmol of heavy labeled
reference peptide was added to the digests, each containing 100 μg of total peptide. After
desalting the peptides with macro-spin columns (Harvard Apparatus), an aliquot containing 1
μg of peptide was subjected to targeted mass spectrometry using previously specified
instrument settings (Picotti et al, 2009 (link)).
The total number of cells in the samples was determined by flow cytometry. Averages and standard
deviations were derived from glucose experiments for the wild-type cells and from glucose and
acetate experiments for other strains.
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