Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

Abstract

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC–MS–MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell–cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids.

Emerging roles of lymphatic endothelium in regulating adaptive immunity

“…Lymph fluid drained from the periphery via afferent lymphatic vessels bathes LNs with soluble foreign antigens as well as tissue-specific self-antigens and any other molecules present in the local periphery, such as cytokines from inflamed tissues or tumors (10). Because lymph originates as interstitial fluid surrounding the cells of peripheral tissues, it is enriched in peptides that are processed extracellularly, including those resulting from local catabolism, ECM degradation, apoptosis, and tissue remodeling (9,11). While tissue-resident DCs take up and process antigens for presentation on MHC molecules, often resulting in DC maturation and migration to LNs (12), free antigens can rapidly drain to the LN via the subcapsular sinus, where large antigens and opsonized material may be directly taken up by subcapsular macrophages (Figure 1).…”

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo

“…Human afferent prenodal lymph samples were collected as described. 22 The lymph was centrifuged at 1500g for 10 minutes to remove all cellular components. All lymph samples were clear at the time of collection, and contaminating hemoglobin levels in samples were measured as less than 1% of that in whole blood.…”

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets

“…40,41 Therefore, although our in vitro and in vivo analyses showed that MMPs are a major form of proteases in degrading core 1 O-glycan-deficient or desialylated PDPN, other proteases such as serine proteases, may also be involved in the regulation of PDPN levels. [42][43][44] PDPN induces platelet aggregation by interacting with CLEC-2 on platelets.…”