Serum prostate-specific antigen (PSA) levels ranging from 4 to 10？ng/mL is considered a diagnostic gray zone for detecting prostate cancer because biopsies reveal no evidence of cancer in 75% of these subjects. Our goal was to discover a new highly specific biomarker for prostate cancer by analyzing plasma proteins using a proteomic technique. Enriched plasma proteins from 25 prostate cancer patients and 15 healthy controls were analyzed using a label-free quantitative shotgun proteomics platform called 2DICAL (2-dimensional image converted analysis of liquid chromatography and mass spectrometry) and candidate biomarkers were searched. Among the 40,678 identified mass spectrum (MS) peaks, 117 peaks significantly differed between prostate cancer patients and healthy controls. Ten peaks matched carbonic anhydrase I (CAI) by tandem MS. Independent immunological assays revealed that plasma CAI levels in 54 prostate cancer patients were significantly higher than those in 60 healthy controls ( , Mann-Whitney test). In the PSA gray-zone group, the discrimination rate of prostate cancer patients increased by considering plasma CAI levels. CAI can potentially serve as a valuable plasma biomarker and the combination of PSA and CAI may have great advantages for diagnosing prostate cancer in patients with gray-zone PSA level. 1. Introduction Prostate cancer is the most common malignancy in the United State. In 2009, 192,280 men were estimated to have been diagnosed with prostate cancer, and 27,360 of these patients died in the United States [1]. The prostate-specific antigen (PSA) is used for the detection of prostate cancer in daily practice, but its diagnostic reliability is hampered by its low specificity. Thus, serum PSA levels ranging from 4 to 10？ng/mL are called the “gray zone” in which it is very difficult to discriminate between patients with prostate cancer and those with benign prostatic hyperplasia (BPH), prostatitis, or normal prostate. Furthermore, among the patients with serum PSA levels between 4 to 10？ng/mL, only 25% will be found to have prostate cancer [2]. Serum PSA levels can also increase in prostatitis, [3, 4] and approximately 20%–30% of prostate cancers are missed when the cut-off value is set to 4？ng/mL [5–7]. The false negative rate in the first biopsy is estimated between 12% and 32% [8, 9], and a large population of men with chronically high serum PSA levels undergo repeated biopsies to eliminate the possibility of prostate cancer [3, 4]. Our quantitative label-free shotgun proteomics analysis system, called 2-dimensional image
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