Reverse stable isotope labelling with Raman spectroscopy for microbial proteomics

Abstract

Global proteome changes in microbes affect the survival and overall production of commercially relevant metabolites through different bioprocesses. The existing methods to monitor proteome level changes are destructive in nature. Stable isotope probing (SIP) coupled with Raman spectroscopy is a relatively new approach for proteome analysis. However, applying this approach for monitoring changes in a large culture volume is not cost‐effective. In this study, for the first time we are presenting a novel method of combining reverse SIP using 13C‐glucose and Deuterium to monitor the proteome changes through Raman spectroscopy. The findings of the study revealed visible changes (blue shifts) in proteome related peaks that can be used for monitoring proteome dynamics, that is, synthesis of nascent amino acids and its turnover with time in a non‐destructive, cost‐effective, and label‐free manner.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging

“…This shift can be readily confirmed, as the positions of unlabelled bands in the cell spectra have been thoroughly investigated in previous studies. 10,21 In one of our recent studies, we reported the utility of this approach for global proteome monitoring. 10 Due to the easy availability and low cost of unlabelled supplements, it is an efficient approach for requirements to working with large growth medium volumes.…”

“…10,21 In one of our recent studies, we reported the utility of this approach for global proteome monitoring. 10 Due to the easy availability and low cost of unlabelled supplements, it is an efficient approach for requirements to working with large growth medium volumes. This offers an attractive opportunity in the future for sensing and visualizing metabolites and metabolic pathways inside cells in situ or in vivo .…”

“…A reverse isotopic effect is observed when the heavier isotope is replaced by the lighter isotope resulting in the reduction of reduced mass and a shift towards a higher wavenumber (blue shift). 9,10…”

Sensing the bactericidal and bacteriostatic antimicrobial mode of action using Raman-Deuterium stable isotope probing (DSIP)

“…This also lead to appearance of other unique biomolecular peaks. 20,21 The appearances of new deuterated peak are the redshifted that arise due to the isotopic effect i.e the substitution by higher mass number D in place of H leading to an increase in the reduced mass of the molecular bond owing to a decrease in wavenumber. [21][22][23] This C-H/D biomolecular substitution occurs due to the rapid intracellular biocatalytic exchange between D of D2O and H atom of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) which is redox active.…”

“…20,21 The appearances of new deuterated peak are the redshifted that arise due to the isotopic effect i.e the substitution by higher mass number D in place of H leading to an increase in the reduced mass of the molecular bond owing to a decrease in wavenumber. [21][22][23] This C-H/D biomolecular substitution occurs due to the rapid intracellular biocatalytic exchange between D of D2O and H atom of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) which is redox active. 19,21 Previously Silvie et al has reported the use of Raman spectroscopy for investigating the action mechanism of bactericidal and bacteriostatic action based on DNA and protein signals in Raman bio fingerprint region (600 -1800 cm -1 ) of Staphylococcus epidermis.…”