The growing usage of nanomaterials is causing emerging concern regarding their environmental behavior in aquatic environments. A major need is the capability to detect and quantify nanomaterials in complex water matrices. Carbon60 fullerene is of special interest because of the widespread application of nanocarbon technology. The present study focuses on how to separate and concentrate fullerenes from water containing salts and organic matter and then quantify their concentrations using liquid chromatography coupled with mass spectrometry (LC/MS). The stable aqueous C60 aggregates (nC60) prepared in the present study were approximately 60 to 70 nm in diameter and had an ultraviolet (UV) extinction coefficient of 0.0263 L/mg-cm at 347 nm, which equated to a UV detection limit of 0.4 mg/L based upon an absorbance of 0.01 cm(-1). Ultraviolet analysis is not applicable to use in waters containing salts or organics (e.g., tap water) because of their interferences and potential to aggregate nC60. The LS/MS analysis detected C60 as single fullerene rather than aggregates. Three techniques were developed to separate and concentrate nC60 from ultrapure and tap water into toluene to facilitate LC/MS determination: Evaporation of sample to dryness; extraction using 20% NaCl into toluene; and solid-phase extraction. The first two methods had limitations for use in complex water matrices, but aqueous nC60 concentration as low as 300 ng/L in water were quantified using solid-phase extraction (SPE) separation method. This is the first publication on the application of extraction methods for nC60 from ultrapure and tap waters and determination of detection limits by LC/MS.

Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.

Osmotic stress promotes production of certain CE proteins and cross-linking membrane-associated TG1 and decreases cell viability via JNK MAPK-mediated pathways. Strategies that inhibit JNK production downregulate the cornification response of PCHCE cells to osmotic stress. These findings have potential therapeutic implications for preventing cornification of the corneal epithelium in response to the hyperosmolar tear film in dry eye disease.

Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50 cm-long column packed with 1.9 μm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of over 300% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results reveal the analytical power of optimized chromatography using sub 2 μm particles for the analysis of the T cell phosphoproteome to reveal a vast landscape of significantly altered phosphorylation changes in response to T cell receptor stimulation.