Figure 1.
BSA oxidation state as a function of the MCO level.
BSA was reacted at increasing MCO levels and analysed by (A) SDS/PAGE (10% polyacrylamide) with Coomassie staining or (B) after DNPH-derivatisation and blotted onto PVDF followed by carbonyl immunostaining. (C) The relative carbonyl content of the BSA monomer using Quantity One densitometry analysis (Biorad) analysis. The carbonyl content of the MCO untreated sample was set to 1.
Figure 2.
Distribution of BSA site-specific oxidation.
BSA is a secreted protein and its precursor contains 607 amino acids (Accession number P02769), whereas the mature form lacks the first 24 residues and contains 583 amino acids. This figure displays the amino acid sequence of the BSA precursor with CS identified by mass spectrometry shaded in gray and oxidised methionines shown by an asterisk (*). Amino acids shown by gray italic characters correspond to regions not found by mass spectrometry analysis (less than 10% of the primary sequence). RKPT-enriched regions (3 carbonylatable sites within a 4 amino acid sequence window) are represented within framed boxes. Thin boxes correspond to RKPT-enriched regions containing at least one CS, whereas thick boxes correspond to RKPT-enriched regions in which no CS were identified. Boxes represented by a dotted line correspond to RKPT-enriched regions in which no CS were identified, but which contain residues that were not covered by mass spectrometry analysis of tryptic peptides.
Table 1.
Identification of specific BSA carbonylated sites.
Table 2.
Mass Spectrometry analysis reveals a reactional pathway of carbonylation.
Table 3.
CS found within the set of 23 E. coli proteins.
Figure 3.
Schematic model of the detection of predicted HSC.
The details of the principle of detection are described in Materials and Methods section. A predicted HSC (grey box) is defined by two regions. A) An RKPT-enriched region (3 carbonylatable residues within a sequence of 4 amino acids (R, K, P, T; 3; 4) containing at least one proline (P; 1; 0). B) A specific environment around an RKPT-enriched region, enriched in various residues: (i) iron binding sites (D, E, Y, H, C, namely 1 residue within a window of 2 residues (D, E, Y, H, C; 1; 2) and 8 residues within a window of 29 residues (D, E, Y, H, C; 8; 29); (ii) hydrophobic amino acids (A, V, G, I, namely 1 residue within a window of 2 (A, V, G, I; 1; 2)); (G namely 2 residues within a window of 14 (G; 2; 14)); and (iii) (P, T) with 2 residues occurring within a window of 21 residues (P, T; 2; 21). E, environment; r, right; l, left and w, window.
Table 4.
Percentage of predicted CP in three organisms.
Figure 4.
Effect of amino acid sequence shuffling of E. coli proteins on predicted HSC occurrence.
We estimated the predicted CP occurrence for each protein of the E. coli proteome after shuffling (see text). A frequency distribution of the obtained values was then generated and plotted either for the set of proteins predicted to be carbonylated (899 proteins, white bars) or for the set of proteins predicted to be non-carbonylated (3364 proteins, black bars) in the E. coli proteome.
Table 5.
Percentages of amino acids and amino acid groups in the E. coli proteome and in the sets of predicted carbonylated and non-carbonylated proteins.
Figure 5.
Analysis of the expression of genes encoding predicted CP.
Frequency distribution of CAI (codon adaptation index) values, within the entire proteome of E. coli (4243 proteins, dark line), predicted CP (899 proteins, gray line) and predicted non-CP (3344 proteins, dotted line). The 95% confidence intervals of the mean were [0.4474–0.4538], [0.4776–0.4915], and [0.4379–0.4451] for the entire proteome, for carbonylated and non-carbonylated proteins, respectively.
Figure 6.
Percentage of predicted CP within functional COG classes.
The predictive CSPD model was run on all protein classes, leading to the determination of the percentage of predicted CP for the standard proteome (Black circles) for each protein class. The average (open circle) and the standard deviations obtained for the 1000 shuffled proteomes (see Materials and Methods section) are indicated for each class. The asterisks highlight significant differences between shuffled and standard proteome in terms of percentages of predicted CP (p value <0.005). Designations of functional categories: C, energy production and conversion, D, cell cycle control and mitosis, E, amino acid metabolism and transport, F, nucleotide metabolism and transport, G, carbohydrate metabolism and transport, H, coenzyme metabolism, I, lipid metabolism, J, translation, K, transcription, L, replication and repair, M, cell wall/membrane/envelope biogenesis, N, cell motility, O, post-translational modification, protein turnover, chaperone functions, P, inorganic ion transport and metabolism, Q, secondary metabolite biosynthesis, transport and catabolism, R, general functional prediction only (typically, prediction of biochemical activity) S, unknown function, T, signal transduction, and U, intracellular trafficking and secretion, V, defense mechanisms. The dotted line represents the percentage of predicted CP for the whole standard E. coli proteome.