Reagent-free LC-MS/MS-based pharmacokinetic quantification of polyhistidine-tagged therapeutic proteins.

AIM: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS. RESULTS: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. CONCLUSION: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.

Contribution of proteomics in the identification of novel proteins associated with plant growth.

The epidermis is not only the interphase between the plant and the environment but also a growth-limiting tissue. Understanding the initiation and regulation of its expansion growth is essential for addressing the need for more food and fuel. We used mass spectrometry to identify proteins from auxin (indole-3-acetic acid; IAA)-induced rapidly growing corn (Zea mays) coleoptiles to find possible candidates controlling this growth as well as the underlying cell wall and cuticle biosynthesis. Excised sections were incubated for 4 h in the absence or presence of IAA, protein extracted, and analyzed using LC-ESI-MS/MS. Of 86 proteins identified, 15 showed a predicted association with cell wall/cuticle biosynthesis or trafficking machinery; four identifications revealed novel proteins of unknown function. In parallel, real-time PCR indicated that the steady-state mRNA levels of genes with a known or predicted role in cell-wall biosynthesis increase upon treatment with auxin. Importantly, genes encoding two of the hypothetical proteins also show higher levels of mRNA; additionally, their gene expression is down-regulated as coleoptile growth ceases and up-regulated in expanding leaves. This suggests a major role of those novel proteins in the regulation of processes related to cell and organ expansion and thus plant growth.

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvß3.

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvß3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA-integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA-integrin binding was specific to sPLA2-IIA and not to the integrin.

Rheumatoid and pyrophosphate arthritis synovial fibroblasts induce osteoclastogenesis independently of RANKL, TNF and IL-6.

Bone destruction is a common feature of inflammatory arthritis and is mediated by osteoclasts, the only specialized cells to carry out bone resorption. Aberrant expression of receptor activator of nuclear factor kappa ß ligand (RANKL), an inducer of osteoclast differentiation has been linked with bone pathology and the synovial fibroblast in rheumatoid arthritis (RA). In this manuscript, we challenge the current concept that an increase in RANKL expression governs osteoclastogenesis and bone destruction in autoimmune arthritis. We isolated human fibroblasts from RA, pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients and analyzed their RANKL/OPG expression profile and the capacity of their secreted factors to induce osteoclastogenesis. We determined a 10-fold increase of RANKL mRNA and protein in fibroblasts isolated from RA relative to PPA and OA patients. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were cultured in the presence of RA, PPA and OA synovial fibroblast conditioned medium. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), F-actin ring formation and bone resorption assays. The formation of TRAP(+), VNR(+) multinucleated cells, capable of F-actin ring formation and lacunar resorption in synovial fibroblast conditioned medium cultures occured in the presence of osteoprotegerin (OPG) a RANKL antagonist. Osteoclasts did not form in these cultures in the absence of macrophage colony stimulating factor (M-CSF). Our data suggest that the conditioned medium of pure synovial fibroblast cultures contain inflammatory mediators that can induce osteoclast formation in human PBMC independently of RANKL. Moreover inhibition of the TNF or IL-6 pathway was not sufficient to abolish osteoclastogenic signals derived from arthritic synovial fibroblasts. Collectively, our data clearly show that alternate osteoclastogenic pathways exist in inflammatory arthritis and place the synovial fibroblast as a key regulatory cell in bone and joint destruction, which is a hallmark of autoimmune arthritis.