Labeling of Bifidobacterium longum Cells with ¹³ C-Substituted Leucine for Quantitative Proteomic Analyses

Abstract

Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [ 13 C 6 ]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence.

Early activation of the fatty acid metabolism pathway by chronic high glucose exposure in rat insulin secretory β‐cells

“…For quantification, MALDIPepQuant (http://www.expasy.org/tools/maldipepquant/) 21, an in‐house software specifically dedicated to SILAC data acquired on a MALDI‐TOF/TOF, was used to quantify the identified proteins. An error of 0.2 Da for doublet detection was allowed, the labels selected were 13 C‐Leu and 13 C 6 / 15 N 2 ‐Lys with respective shifts of 6 and 8 Da on amino acids L and K. Echo peaks were included for peptides containing at least three labeled amino acids.…”

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

“…2D electrophoresis-based workflows, the most popular techniques for the study of bacterial stress responses, do not provide information on hydrophobic proteins, mainly membrane proteins, due to technical difficulties in retaining their solubility during the isoelectrofocusing and limitations in reliable quantification (Bunai & Yamane, 2005;Speers & Wu, 2007). Some of these disadvantages have been overcome with new techniques based on improved solubilizing agents or isotopic labelling (Couté et al, 2007;Speers & Wu, 2007), expanding the possibilities for analysing bacterial membrane subproteomes. In fact, SILAC has recently been successfully applied to the monitoring of changes in the membrane proteome during stationary-phase adaptation of Bacillus subtilis (Dreisbach et al, 2008).…”

“…Proteins were considered as identified if they contain at least one peptide with score and P-value above respectively 6.0 and 1610 24 . The SILAC ratio was determined by averaging the isotope cluster areas of heavy and light peptides identified for each protein using MALDIPepQuant, an inhouse software specifically developed for SILAC data acquired on a MALDI-TOF/TOF instrument (Couté et al, 2007). To evaluate the effect of bile the mean of the SILAC ratios from the three independent batches was considered.…”

“…Each experiment was designed for a pair of cultures: a control culture (non-bile-containing SDMBL broth) and a bile-containing culture. In each pair, one of the cultures was grown with normal leucine and the other with [6-13 C]leucine, as previously described in detail by Couté et al (2007). Membrane extracts for each pair were mixed at theoretical protein concentration ratios of 1 : 1, and 30 mg of the mix was run on SDS-PAGE gels.…”

“…The inoculum was obtained by culturing the strain on MRS agar plates (BD Diagnostic Systems) supplemented with 0.05 % (w/v) L-cysteine (Merck) (MRSC), and subsequently an isolated colony was transferred to MRSC broth and grown overnight. The inoculum was washed three times with semidefined medium for B. longum (SDMBL, Couté et al, 2007), resuspended in this medium, and diluted appropriately in 1 litre of SDMBL [containing a sublethal concentration of bile salts, 0.6 g l 21 (Ox bile extract LS55; Oxoid), when needed] to allow B. longum to grow for 13 generations and the harvesting of the cells in the exponential phase of growth (OD 600 0.4-0.8), as previously described (Couté et al, 2007). For SILAC analysis of proteins from the membrane fraction, one of the cultures (control or bile-containing culture, depending on the pair) was grown in the presence of [6-13 C]leucine.…”