Label-free Quantitative Expression Profiling of Liver Proteins from Fathead Minnows Field- Exposed to Waste Water Treatment Plant Effluent

摘要

1. The most commonly employed MS-based approach for quantitative protein expression profiling directly from animal model sinvolve two-dimension algel electrophoresis followed by differential spot detection and LC-MS. In addition to having alimited dynamicrange in detecting quantitative differences and the ambiguity of multiprotein signal contributions within any one spot, the technique reliesoncalculating aratioagainst acommon sample rather than directly acrossall samples, thus limiting the throughput and the number of unique samples that can be directly compared. 2. Here we apply analternative, area-under-the-curvelabel-free LC-MS based approach to quantitatively characterize protein expression profiles directly from hepatict issue (whole liver) homogenates off athead minnows(Pimephalespromelas) exposed to dechlorinated tapwater(n=4) or effluent from a was tewater treatment plant(WWTP)(n=6) in Kiawah Island, SC for 14 days. 3. Acrossall animals, a total of 1084 unique proteins (4528 unique peptides) were identified at a 1.0% FDR from asingledimension rp-LC separation of 1ugtotaldigeston column. Analytical variation of yeast alcohol dehydrogenase proteinspikes across all samples was 9.9%(RSD) and the averageanalytical variation from all 1084 proteins across replicate QC pools run every five injections was 8.2%(RSD). 4. Error weighted ANOVA statistical analysis identified 55 proteins which were up-regulated and 24 proteins which were down-regulated (> 2-fold, p-value <0.05) as a function of WWTP exposure. Ingenuity pathway analysis of differentially expressed proteins indicated dysregulation in cellular function and maintenanceproteins including ABP1, adrug binding molecule which controls the level of putrescine and histamine in tissues, as wellas inflammatory response pathway proteins including ADABP, a receptor involved in the costimulatory signal essential for T-cell activation.