The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics. Therefore, it is necessary to define the minimum number of individuals required for sampling to represent the normal urinary proteome.

Background: Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde cross-linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can Fish out complexes that include particular target proteins, but affinity-based co-purification has a limited capacity to eliminate nonspecific binding to beads and/or antibodies. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. Results: We described a 4 F strategy to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation: Fast Fix, Fish, and Filter, using albumin interactome as an example. The use of gel excision without staining makes this strategy comprehensive and sensitive. The target protein must be identified in the same slice as its ligands. The ligands must be identified in slices for the experimental group but not in the corresponding control slices. Only proteins that appear in the range of molecular weights equal to or greater than the sum of the proteins’ theoretical molecular weights, together with the target, are considered ligands. In this study, 5 s of cross-linking with 10% formaldehyde was achieved in human blood. The use of this strategy identified 35 ligands for albumin. Comparison with four major previous studies of the albuminome revealed that 68.57% of the 35 ligands identified in our study were identified in these other studies. Conclusions: Fast cross-linking was achieved. The 4 F strategy can be used to identify real-time in situ interactions without prior intervention and to comprehensively identify ligands of particular target proteins with fewer false positives.

Background: Liver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases.Results: In this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma.Conclusions: These results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.

Serum proteins carry out several functions in the circulation, including transfer, immunological functions, messenger functions, coagulation, and regulation of homeostasis. To investigate changes in serum proteins that occur during development, the serum proteomes of embryonic 15.5 (E15.5) fetuses and newborn rats were compared using LC-MS/MS. A total of 958 proteins were identified in the serum of rats at both developmental stages. The serum proteome pattern of newborn rats was compared to E15.5 fetuses by relative quantitation. The expression patterns of hemoglobin subunits were different at the two stages, with most of the subunits having decreased expression in newborn rats compared to E15.5 fetuses. In addition, 8 of 12 apolipoproteins were significantly decreased and 10 of 11 identified complement molecules were increased, with 4 exhibiting a significant increase. Moreover, 11 of 14 of the significantly increased enzyme regulators were inhibitors. The serum proteome patterns of different littermates from both developmental stages were also compared. We found that the levels of many highly abundant serum proteins varied between littermates, and the variations were larger than the variations of the technical control. The serum proteomes of newborn rats and E15.5 fetuses were compared. The expression patterns of hemoglobin subunits were different at the two developmental stages, with most of the subunits having decreased expression. The majority of apolipoproteins had significantly decreased expression, while almost all identified complement proteins had increased expression. The levels of several highly abundant serum proteins also varied among littermates at these two developmental stages. This is the first study using LC-MS/MS to investigate serum proteome development.

It remains a challenge to endow a polymeric material with antithrombotic ability by surface grafting without disturbing the bulk properties of the substrate. Heparin-based functional structures of less than 80 nm were fabricated and covalently grafted on a polyethylene terephthalate surface via carbene chemistry (Hep-g-PET). Heparin was oxidized with the minimum antithrombrin sequence retained, creating an aldehyde group on the chain terminus. Oxidized heparin was then covalently attached to a poly(amidoamine) (PAMAM)-grafted PET substrate. The interface between blood and PET was improved by the surface functionality, and the amount of attached platelets decreased to 29 ± 12.1% of its initial value. The bulk properties of the functionalized film were hardly influenced, and the visible light transmittance remained more than 96%. The tethered structures also showed the ability to kill attached S. aureus and E. coli efficiently. The functionalized membrane showed negligible ex vivo cell cytotoxicity and a low hemolysis ratio. Hep-g-PET was implanted in between rat skin and muscle, and showed an outstanding histological response and antimicrobial ability. The influences of the graft thickness and the heparin chain length were explored. The strategies reported in this work may help to improve the design of polymeric implant bio-devices.