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Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and Gamma-globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89-0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85-0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

Background: We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development. Methods/Principal Findings: Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs (+AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to +AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells. Conclusion: We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer. [ABSTRACT FROM AUTHOR]

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-β (Aβ) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer' s disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Aβ with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Aβ30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of ±4385 Da determined by electrospray ionization mass spectrometry. This diamine-and gadolinium-substituted derivative of Aβ is shown to have enhanced in vitro binding to Alzheimer' s disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PSl transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed. [ABSTRACT FROM AUTHOR]