Bottom-Up Proteomics Map Healthy Human Lymph and Plasma Protei…

The use of bottom-up proteomics for the mapping of protein profiles in compartments of cells, in differentiated tissue, or in whole organisms is an established technique to understand protein expression and quantification of protein levels. Using this knowledge of expression and quantification aids in the establishment of tissue- or disease-specific biomarkers for diagnostic testing. Biomarkers can then be used to quickly evaluate the state, diseased or otherwise, of a tissue, cell, or organism being tested. The characterization of the proteome content of lymph provides unique difficulties because of the isolation deep within blood vessels, the continuously changing protein composition, and the eventual remixing with the blood plasma. Lymph is a bodiliy fluid composed of the fluids from the interstitial space from various tissues. The fluid is collected through devoted capillaries surrounding tissues comprised of various metabolic products and enzymes. The lymph is collected via vessels and lymph nodes and recirculated into the blood via subclavian veins, where is it combined with blood plasma.¹

Since the lymph is mixed back into the blood, the prevailing hypothesis was that there would be nearly 100% overlap between the proteomes of prenodal afferent lymph and plasma itself. But additional protein identifications and efforts at proteomic mapping of these two different fluids has caused this hypothesis to undergo recent scrutiny with the identification of unique protein identifications in one of the fluids or another.¹ In Clement et al.,² the baseline proteome of afferent pernodal lymph and plasmid from healthy patients was measured. Using a combination of techniques, 253 proteins were identified in afferent pernodal lymph and plasma. Two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) was used for rough quantification of protein levels to measure differences in expression through up- or downregulation of specific proteins common to both fluids. Using one-dimensional gel electrophoresis nanoliquid chromatography-tandem mass spectrometry on an Orbitrap and LTQ mass spectrometer (Thermo Scientific), 144 of the 253 identified proteins were shown to be common between the lymph and the plasma. The remaining 109 proteins were unique to either the lymph (72 proteins) or the plasma (37 proteins) specifically. Interestingly, the identifications of the unique proteins for each of the biological fluids provides mechanistic insight into both their origins and their functions.

Of the 144 proteins found to be common between the plasma and the lymph, they were primarily components of complement activation and blood coagulation, transporters, and protease inhibitors. The lymph-specific protein identifications were skewed toward intracellular proteins. A large portion of the lymph-specific proteins could be classified as being part of the “connective tissue development and function” functional proteomic classification group as established by functional network analysis (IPA analysis). An interesting enrichment found in the lymph fractions consisted of nuclear proteins, such as histones, splicing, and transcription factors. In direct contrast to lymph-enriched proteins, plasma-specific proteins were, in majority, extracellular and membrane-bound proteins and were not enriched for intracellular and nuclear proteins. The unique proteins associated with plasma mostly were involved in lipid transport and metabolism.

The ultimate goal of comparative proteomic studies of differentiated tissue, including healthy tissue, is to gain a greater understanding of the basal patterns of expression in these sub-populations of cells and tissues. Bottom-up proteomics studies such as Clement et al.², provide valuable insight into specific expression patterns for hundreds of proteins in two unique but associated biological fluids. The results presented further bolster the hypothesis that lymph is more than an ultrafiltrate of blood plasma but rather a unique biological fluid with a specific immunological role and function. The identification and mapping of proteomic profiles of pathways and systems from healthy individuals is an important step in establishing a baseline for future proteomic studies in the search for disease relevant biomarkers.

References

1. Leak, L.V., et al. (2004) ‘Proteomic analysis of lymph‘, Proteomics, 4 (3), (pp. 753-765)

2. Clement, C.C., et al. (2013) ‘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‘, Journal of Proteomics, 78 (1), (pp. 172-187)

3. Clement, C.C., et al. (2010) ‘An expanded self-antigen peptidome is carried by the human lymph as compared to the plasma‘, PLoS One, 5 (3), (pp. 1-10)