Plasma proteome response to severe burn injury revealed by 18O-labeled "universal" reference-based quantitative proteomics

Abstract

A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of approximately 35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

Figure 1. Flowchart depicting the overall experimental strategy

Control and burn patient plasma samples along with a pooled reference sample are subjected to initial IgY12 depletion followed by trypsin digestion. The reference digest is labeled with ¹⁸O, and then an equal amount of labeled reference is mixed with each patient sample to generate individual samples spiked with the ¹⁸O reference. The spiked samples are further fractionated using a cysteinyl-peptide enrichment strategy prior to LC-MS analysis.

Figure 2. Reproducibility between replicate analyses performed more than three months apart

(A) Scatter plot for peptide Log₂Ratio data between the two replicates of a single sample. (B) Histogram of the distribution of coefficients of variation (CV) for all peptide Log₂Ratio data. CV values were calculated based on the standard deviations of the abundance ratio data between the two replicate analyses relative to the average abundance ratio for each peptide. (C) and (D) Protein abundance profile comparisons between the two replicate data for two selected proteins.

Figure 3. Histogram of p-value distribution

P-values are based on the statistical ANOVA test for all quantified proteins across the three biological groups (control, burn time-point 1, burn time-point 2).

Figure 4. Heatmap of protein abundances across the 40 control and burn patient plasma samples

Only statistically significant proteins are displayed. Each row represents an individual protein, and each column represents a biological sample. All abundances are displayed in Log₂Ratio format relative to the labeled reference sample. Two blank columns were inserted to separate the three biological groups.

Figure 5. Protein abundance changes for selected proteins in bar graphs

Standard deviations were shown on each of the bars. Burn-T1 indicates sample collection at hospital admission and Burn-T2 indicates sample collection at a peak MOF episode. The differences between controls and both burn time-points are significant for all proteins (p<0.02).

Figure 6. Pathways mapped by proteins with significant abundance changes

(A) Top 5 canonical pathways based on the number of proteins mapped. (B) A partial representation of acute phase signaling pathways. The green color of mapped proteins indicates a decreased concentration observed following burn injury.