BACKGROUND: Autologous blood products, such as platelet-rich plasma (PRP) are commercial products broadly used to accelerate healing of tissues after injuries. However, their content is not standardized and significantly varies in composition, which may lead to differences in clinical efficacy. Also, the underlying molecular mechanisms for therapeutic effects are not well understood. PURPOSE: A proteomic study was performed to compare the composition of low leukocyte PRP, platelet poor plasma (PPP), and blood plasma. Pathway analysis of the proteomic data was performed to evaluate differences between plasma formulations at the molecular level. Low abundance regulatory proteins in plasma were identified and quantified as well as cellular pathways regulated by those proteins. METHODS: Quantitative proteomic analysis, using multiplexed isotopically labeled tags (TMT labeling) and label-free tandem mass spectrometry, was performed on plasma, low leukocyte PRP, and PPP. Plasma formulations were derived from two blood donors (one donor per experiment). Pathway analysis of the proteomic data identified the major differences between formulations. RESULTS: Nearly 600 proteins were detected in three types of blood plasma formulations in two experiments. Identified proteins showed more than 50% overlap between plasma formulations. Detected proteins represented more than 100 canonical pathways, as was identified by pathway analysis. The major pathways and regulatory molecules were linked to inflammation. CONCLUSION: Three types of plasma formulations were compared in two proteomic experiments. The most represented pathways, such as Acute Phase Response, Coagulation, or System of the Complement, had many proteins in common in both experiments. In both experiments plasma sample sets had the same direction of biochemical pathway changes: up- or down-regulation. The most represented biochemical pathways are linked to inflammation.

Endometriosis, ectopic growth of the uterine lining (endometrium), which affects 6-11% of reproductive age women, is associated with pelvic pain and infertility. We investigated the peritoneal fluid (PF), urine and omental fat (OF) proteomes of women with endometriosis vs. individuals with no surgically visualized endometriosis. All participants were enrolled in the NICHD-funded ENDO Study. A two-step proteomic study was performed. The first, a broad survey, employed a semi-quantitative gel LC-mass spectrometry (MS) workflow: SDS PAGE fractionation, trypsin digestion and LC-MS/MS. The results showed sample integrity but failed to detect any differences between women with and without endometriosis. The second step was a quantitative analysis of OF samples. We employed another sample set (n=30) from women ± disease and isobaric mass-tag (iTRAQ) chemistry to label peptides and 2D LC-MS/MS for protein identification and quantification. Three proteins-matrix metalloproteinase-9, neutrophil elastase, and FAM49B-were significantly lower in abundance in samples from women with endometriosis. Interestingly, neutrophil elastase and FAM49B levels were associated with higher levels of a subset of endocrine disrupting chemicals (EDCs) that were previously measured in the same samples. The results of these experiments showed the feasibility of associating endometriosis with changes in the OF protein repertoire and EDC levels.

Biological significance

Endometriosis, pathological growth of the uterine lining, is associated with significant morbidities, including pain and infertility. However, the causes of this common condition are poorly understood. This study determined whether endometriosis was associated with changes in the protein composition of peritoneal fluid, urine and/or omental fat. A protein of unknown function (FAM49B) and two proteinases (metalloproteinase-9, neutrophil elastase) were down regulated in OF samples from women with versus without endometriosis. These findings suggested proteinase imbalances at sites that were distant from the endometriotic lesions. Additionally, FAM49B and neutrophil elastase levels were associated with higher levels of a subset of environmental chemicals that were quantified in the same samples, suggesting other possible associations. Thus, this work generated hypotheses that will be tested in further studies.