Lymph is not a plasma ultrafiltrate: a proteomic analysis of injured patients

Abstract

Studies on animal models have documented a role for the water-soluble protein fraction of mesenteric lymph as a conduit from hemorrhagic shock to acute lung injury and postinjury multiple organ failure. We hypothesize that mesenteric lymph is not an ultrafiltrate of plasma and contains specific protein mediators that may predispose patients to acute lung injury/multiple organ failure. Mesenteric lymph and plasma were collected from critically ill or injured patients and from nine patients with lymphatic injuries, during semielective spine reconstruction, or immediately before organ donation. Proteomic analyses were performed through immunoaffinity depletion of the 14 most abundant plasma proteins and 1D gel electrophoresis followed by liquid chromatography coupled online with mass spectrometry analyses. Overall, 548 proteins were identified in the patients undergoing semielective surgery, of which 155 were uniquely present in the lymph. In addition, the postshock plasma proteome was characterized by peculiar features, suggesting that only a partial overlap exists between the plasma and mesenteric lymph from trauma patients. Differential proteins between the matched plasma and mesenteric lymph from trauma patients could be related to coagulopathy and hypercoagulability, cell lysis, proinflammatory responses and immune system activation, extracellular matrix remodeling, lymph-specific immunomodulation and vascular hypoactivity/neoangiogenesis, and energy/redox metabolic adaptation to trauma. In conclusion, the proteome of mesenteric lymph is biologically different (in qualitative and quantitative terms) than that of a mere plasma ultrafiltrate.

Figure 1

Scaffold report of proteome changes in post-shock mesenteric lymph and plasma, as gleaned via HPLC-MS/MS label free peptidomics approaches on nine paired biological replicates per group. In A, a Venn diagram indicates the shared features (232), and those differentially represented only in mesenteric lymph (105) or plasma (23). In B, GO term enrichment for biological functions of the total list of 360 proteins. In C and D, GO term enrichment for cell compartment and biological functions for the 105 features unique to post shock mesenteric lymph, respectively.

Figure 2

Hierarchical clustering analyses of quantitative dynamic changes of post-shock mesenteric lymph and plasma proteins, either increasing in the former group (A), or in the latter (B), as gleaned by label free quantitative proteomics approaches on paired samples from 9 patients undergoing semi-elective spine injury reconstruction. Quantitative emPAI values were obtained through Mascot search and exported from Scaffold into xls file for heat mapping and hierarchical clustering analysis (Pearson correlation-1) through the software GENE-E. Values increase progressively from blue to red in the color scale, as indicated in the legends on top of each cluster. Proteins are enlisted with their relative UniProt ID names, though the _HUMAN taxonomy specification for Homo sapiens has been deleted as to improve the clarity of the figure. Further details are reported in Table 2 (protein list).

Figure 3

Hierarchical clustering analyses of quantitative dynamic changes of post-shock mesenteric lymph and plasma proteins, either increasing in the upper cluster, or decreasing in the lower on, as a result of a Mascot search including semitryptic peptides and at least one peptide (>95% probability, Mascot score>30) for a positive identification. Only proteins non-redundant with Figure 1 were plotted. Quantitative information was gleaned through label free quantitative proteomics analyses on paired samples from 9 patients undergoing semi-elective spine injury reconstruction. Quantitative emPAI values were obtained through Mascot search and exported from Scaffold into xls file for heat mapping and hierarchical clustering analysis (Pearson correlation-1) through the software GENE-E. Values increase progressively from blue to red in the color scale, as indicated in the legends on top of each cluster. Proteins are enlisted with their relative UniProt ID names, though the _HUMAN taxonomy specification for Homo sapiens has been deleted as to improve the clarity of the figure. Further details are reported in Table 3 (protein list).