title: Proteomic analysis of cisplatin-induced cochlear damage: methods and early changes in protein expression.
abstract: 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.
authors.affiliation: Center for Hearing and Deafness, 137 Cary Hall, University at Buffalo, Buffalo, NY 14214, USA. dcoling@buffalo.edu
authors.forename: Donald E
authors.initials: DE
authors.lastname: Coling
authors.forename: Dalian
authors.initials: D
authors.lastname: Ding
authors.forename: Rebeccah
authors.initials: R
authors.lastname: Young
authors.forename: Maciej
authors.initials: M
authors.lastname: Lis
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Stofko
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Blumenthal
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Salvi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002945:Cisplatin / Q000633:toxicity*; D003051:Cochlea / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000378:metabolism; D007526:Isoelectric Point; D018832:Molecular Chaperones / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D010449:Peptide Mapping; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D011916:Rats, Inbred F344; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D046509:Tissue Culture Techniques; D000091342:Endoplasmic Reticulum Chaperone BiP
chemical_list: D000970:Antineoplastic Agents; D002135:Calcium-Binding Proteins; D002945:Cisplatin; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011506:Proteins; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C000630652:Nucb1 protein, rat