Proteomic analysis of cisplatin-induced cochlear damage: methods and early changes in protein expression.

To identify early changes in protein expression associated with cisplatin ototoxicity, we used two dimensional-difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometry to analyze proteins from P3 rat cochleae that were cultured for 3h with or without 1mM cisplatin. Replicate analysis of fluorescent images from six gels revealed significant (p<0.01) cisplatin-induced changes (greater than 1.5-fold) in expression of 22 cochlear proteins. These include increases in the expression of five proteins, four of which were identified as nucleobindin 1, a nuclear calcium signaling and homeostasis protein (2.1-fold), heterogeneous nuclear ribonucleoprotein C, an RNA processing protein (1.8-fold), a 55 kDa protein that is either endothelial differentiation-related factor 1 or alpha-6 tubulin (1.7-fold), and calreticulin, a calcium binding chaperone of the endoplasmic reticulum (ER, 1.6-fold). The expression of 17 proteins was significantly (p<0.01) decreased by greater than 1.5-fold. These include ribonuclease/angiogenin inhibitor 1 (1.6-fold), RAS-like, family 12 (predicted), ras association (RalGDS/AF-6) domain family 5 (4.5-fold), homologous the RAS family of GTPase signaling proteins (2.4-fold), and Protein tyrosine phosphatase domain containing 1 (predicted, 6.1-fold). We identified seven cochlear proteins with either smaller (1.2-1.5-fold) or less significant (p<0.05) cisplatin-induced changes in expression. Notably, heat shock 70 kDa protein 5 (Hspa5, Grp78, and BiP), an ER chaperone protein involved in stress response, decreased 1.7-fold. We observed changes consistent with phosphorylation in the level of isoforms of another ER stress-induced protein, glucose-regulated protein Grp58. Changes in cisplatin-induced protein expression are discussed with respect to known or hypothesized functions of the identified proteins.

Glycerol concentrations required for the successful vitrification of cocktail conditions in a high-throughput crystallization screen.

The Hauptman-Woodward Medical Research Institute runs a high-throughput crystallization screening service in which macromolecules are screened against 1536 potential crystallization cocktails. Typically, multiple crystallization leads are identified. With a limited amount of sample, the question becomes ;How many leads can be optimized and which leads are most likely to produce X-ray diffraction data?'. In order to prioritize the hits for optimization, the amount of glycerol required to successfully cryocool each cocktail has been determined for the cocktails used in the high-throughput screen. Those hit conditions that require the minimum amount of cryoprotectant for successful vitrification will be closer in chemical make-up to the mother liquor. Hence, if the physical properties of the crystals are similar, one could logically prioritize leads that are more likely to produce diffraction based upon the chemical similarity of the native to the cryopreserved mother liquor.