2007
DOI: 10.1128/aem.00667-07
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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.

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Cited by 11 publications
(13 citation statements)
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“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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).…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
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