Klee Eric W, Bondar Olga P, Goodmanson Marcia K, Trushin Sergey A, Bergstralh Eric J, Singh Ravinder J, Anderson N Leigh, Klee George G
From the Departments of Health Sciences Research (Dr E. Klee and Mr Bergstralh) and Laboratory Medicine and Pathology (Drs Bondar, Trushin, Singh, and G. Klee and Ms. Goodmanson), Mayo Clinic College of Medicine, Rochester, Minnesota; and the Plasma Proteome Institute, Washington, DC (Dr Anderson).
Arch Pathol Lab Med. 2014 Oct;138(10):1381-6. doi: 10.5858/arpa.2013-0462-OA.
Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays.
To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays.
Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer.
The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis.
This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.
前列腺特异性抗原（PSA）是一种具有胰凝乳蛋白酶样酶活性的34 kDa糖蛋白，以游离形式和与包括抗胰凝乳蛋白酶和α2-巨球蛋白在内的各种酶抑制剂结合的形式循环。与α2-巨球蛋白结合的前列腺特异性抗原不能被商业PSA免疫测定法检测到。
开发一种质谱测定法，该方法能检测与免疫测定法相同形式的PSA，可作为统一PSA免疫测定法的参考。
从血清中免疫提取前列腺特异性抗原，用胰蛋白酶消化，然后在API 5000光谱仪上对LSEPAELTDAVK肽进行定量。通过向女性血清中添加10%的游离PSA和90%与抗胰凝乳蛋白酶结合的PSA来制备校准品。该测定法根据世界卫生组织96/670参考标准进行标准化。使用来自100名接受前列腺活检的男性（50名阴性，50名患有癌症）的冷冻血清等分试样以及从5名晚期前列腺癌男性随时间收集的5份系列样本，进行临床实用性验证并与2种免疫测定法（罗氏cobas和贝克曼Access）进行比较。
抗体提取效率大于99%。该测定法的分析范围为1.2至76 ng/mL，精密度在1.5 ng/mL时为8.6%，在27 ng/mL时为5.4%。质谱测定法与2种免疫测定法相关性良好。使用受试者工作特征曲线分析，所有3种测定法在区分前列腺癌和良性疾病方面均显示出统计学上的等效性。
这种质谱测定法能够可靠地测量人血清中的PSA浓度，可作为统一PSA免疫测定法的参考标准。
