Cancer-specific protein biomarkers have great potential for informing early detection, diagnosis, and prognosis, as well as monitoring disease progression, response to treatment and therapy, and detection of early recurrence1–4
. One of the most familiar examples is the development and subsequent routine application of measuring prostate-specific antigen (PSA, also known as human kallikrein 3, hKLK3), a protein biomarker that has revolutionized the management of prostate cancer5
. The American Cancer Society has recommended routine use of the total PSA test for early detection of prostate cancer, in combination with digital rectal exam, for males age 50 and older6, 7
. However, the increase in early detection of prostate cancer achieved via application of routine PSA testing has also introduced clinical challenges, particularly in over-diagnosis and in the stratification of risk for subsequent development of highly invasive prostate cancer8, 9
. The inability of current PSA tests to distinguish between indolent and aggressive disease has prompted a recent draft guideline from the US Preventive Service Task Force recommending against PSA screening for anyone without obvious symptoms of prostate cancer7
. Thus, there have been numerous efforts to refine PSA assays to improve the discriminatory power of the test.
One such approach to increase the specificity of PSA screening exploits the biochemical characteristics of PSA. PSA is a 237-amino acid, single chain, serine protease, synthesized in the ductal epithelium and prostatic acini and secreted as a glycoprotein into the lumina of the prostatic ducts. In blood there are two mature forms of PSA, free and bound. The bound form is primarily complexed with protease inhibitors, such as alpha-1- antichymotrypsin (ACT) or alpha-2-macroglobulin (A2M). Even though the molar concentration of ACT and A2M are 1,000-fold higher than that of PSA, up to 45% of PSA in blood is still in an unbound or free form. Clinical assays for ‘total PSA’ do not discriminate between the complexed and the free forms of PSA. However, since A2M engulfs the PSA molecule and blocks the access of anti-PSA antibodies, the ‘total PSA’ assay measures essentially free PSA and the PSA-ACT complex.
Although a total serum PSA concentration of ≥4.0 ng/mL is typically used as an indication for prostate biopsy10
, results from the Prostate Cancer Prevention Trial indicated that up to 27% of men with total PSA concentrations between 3.1–4.0 ng/mL have prostate cancer and that there is a significant risk of detection for prostate cancer over all PSA concentrations11, 12
. At a total PSA concentrations exceeding 10 ng/mL, approximately 50% of the patients had cancer1, 5
. At the intermediate range of total PSA concentrations, 4.0–10.0 ng/mL, 25 – 35% of patients had cancer based on biopsy1
. In an attempt to improve the predictive power of PSA assays, assays have been developed that discriminate between ‘free’ (uncomplexed) and ‘total’ (free + ACT-bound) PSA. Determination of the percent free PSA (%fPSA = free PSA/total PSA ×100) is recommended for risk assessment of patients with total PSA concentrations between 4–10 ng/mL. A %fPSA of >25% indicates a low risk of cancer (e.g., probability = 8%) whereas a %fPSA of <10% suggests a high risk (e.g., probability = 56%)13–15
. A cut-off value of 25% for %fPSA detected 95% of cancers and reduced the biopsy rate by 20% when total PSA levels were between 4–10 ng/mL13
While commercially available immunoassays for both total and free PSA work well and have FDA approval, the development of these assays required the identification and characterization of immunoassay-qualified antibodies that could accurately and reproducibly discriminate between the two biochemical forms of PSA. The investment in time and resources required to generate such immunoassays is considerable, and this requirement often impedes the development of clinically useful protein-based assays in the absence of compelling pre-clinical data. A case in point is the fusion transcript TMPRSS2-ERG, which is present in approximately 50% of prostate cancers16–18
. The fusion transcript is expressed as a truncated protein19, 20
. However, no commercial immunoassays to detect the truncated ERG as a cancer biomarker have been developed.
The focus of this report is the development and evaluation of an alternative approach that circumvents the necessity to develop affinity reagents, known as selected reaction monitoring (SRM) or multiple reaction monitoring21
. SRM exploits the unique features of the triple quadrupole mass spectrometer that enable two levels of mass selection (precursor and fragments) and a relatively long dwell time over a narrow m/z
range of interest, resulting in high selectivity and sensitivity. A further analytical requirement, identical liquid chromatography (LC) elution times for multiple transitions of the same target analyte, filters out the co-eluting background ions with great effectiveness, even from an extremely complex biological matrix, e.g., tryptic digest of plasma. The ion currents of fragment ions can provide accurate quantification of analyte concentration with stable isotope-labeled internal standards. Applying modern triple quadrupole mass spectrometers with high-duty cycles and “smart” SRM assay configurations (e.g., utilizing the peptide LC elution time to “schedule” SRM events), a large number of protein targets can be monitored during a single LC-SRM-MS analysis. These features, combined with several front-end enrichment methods that have been recently developed, e.g., major serum/plasma protein depletion alone, or in conjunction with chemical22
, and antibody-based24
enrichment, have led to reliable detection of targeted proteins at the low ng/mL level or better in serum/plasma25
Published detection limits for total PSA in plasma/serum using SRM-MS are in the 1–10 ng/mL range, and are highly dependent on the sample preparation and the MS detection methods used23, 26–28
. In this study, we describe simple, yet effective, immunoaffinity depletion-based workflows and demonstrate for the first time the detection of both the total and free PSA at the low ng/mL concentration in human clinical serum samples, using LC-SRM-MS without the requirement for specific PSA antibodies. Furthermore, the correlation observed between clinically approved immunoassay tests and SRM-based assays for both the total and free PSA measurements exceeded 0.90, even in a set of blinded samples. This LC-SRM-MS approach can obviously be extended to the quantitative analysis of many other biomarkers that have similar bound and free interactions. More generally, the strong correlations obtained between the LC-SRM-MS analyses and clinical immunoassays suggest that SRM can be used as a reference method for preliminary determination of assay validity, prior to the development of more conventional immunoaffinity-based assays that would be used in clinical laboratories.