Differential proteomic analysis using isotope‐coded protein‐labeling strategies: Comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34

Abstract

Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed.

Modelled microgravity cultivation modulates N-acylhomoserine lactone production in Rhodospirillum rubrum S1H independently of cell density

“…The RPM has been extensively used to study cytoskeleton structure and motility of human cells (Meloni et al, 2006;Walther et al, 1998) and plant gravitropism (Barjaktarović et al, 2009;Hoson et al, 1997) and more recently the RPM has been used to study bacterial cultivation (Beuls et al, 2009;Crabbé et al, 2010;Leroy et al, 2010;Mastroleo et al, 2009b;Mauclaire & Egli, 2010). In the present study, at the proteomic level, only a few common proteins were found in the response of R. rubrum S1H to LSMMG and RPM cultivation, and the LSMMG appeared to induce a higher number of significantly regulated proteins than the RPM compared to the control conditions.…”

“…As mentioned above, the increased AHL production independent of cell density suggests that the response of the bacterium R. rubrum S1H to LSMMG was related to the fluid quiescence and reduced mixing conditions. The latter was also hypothesized for Escherichia coli K-12 cultivated in LSMMG (Tucker et al, 2007;Vukanti et al, 2008) and Cupriavidus metallidurans CH34 cultivated in RPM (Leroy et al, 2010). However, membrane stress response and mechanosensitive-related gene induction among others indicate that a direct mechanical effect on the R. rubrum cell membrane cannot be fully excluded.…”

A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16

“…After 6 h, when 50% of linuron was degraded and an OD 600 of 0.5 was reached, the cultures were centrifuged (3,400 ϫ g, 15 min, 4°C), and the pellets were stored at Ϫ80°C for protein extraction. Protein extraction, post-digest ICPL and MuDPIT (MultiDimensional Protein Identification Tool) were performed as described previously (27). For each experimental condition, duplicate cultures were analyzed.…”

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

“…These disadvantages limit the application of the ICPL labeling method. [58] mTRAQ is another amine-specific nonisobaric labeling technology. Peptides labeled with mTRAQ reagents have identical retention time and ionization efficiency, but different masses (0, 4 or 8 Da for arginine-containing peptides, and 0, 8 or 16 for lysine-containing peptides for triplex labeling).…”