Quantitative Analysis of Prostate Specific Antigen Isoforms Using Immunoprecipitation and Stable Isotope Labeling Mass Spectrometry

Abstract

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

“…Targeted mass spectrometry (MS) is a powerful alternative to Western blot and enzyme-linked immunosorbent assay (ELISA) for quantification of proteins. It provides accurate quantification, high level of specificity, avoiding interference due to cross-reactivity of antibodies, and the ability to discriminate between isoforms (Chen D. et al, 2015; Chen Y.-T. et al, 2015; Jedrychowski et al, 2015; Lesur et al, 2015). Nevertheless, MS-based detection of low-abundant proteins in complex fluids or tissues remains challenging without efficient sample preparation protocols.…”

“…Nevertheless, MS-based detection of low-abundant proteins in complex fluids or tissues remains challenging without efficient sample preparation protocols. The gold standard relies on the combination with immunoprecipitation to selectively enrich the analyte of interest prior to MS (Chen Y.-T. et al, 2015), but is applicable only when antibodies with sufficient specificity and affinity for the target protein are available.…”

Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study

“…To overcome this, studies have been utilising immunoprecipitation (IP) MS to ‘extract' PSA for MS analysis. Utilising stable isotope labelling-multiple reaction monitoring MS (SIL/MRM-MS), it has been possible for one group to simultaneously measure multiple biomarkers including various PSA forms (Chen et al , 2015). …”

Liquid‐phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2014–2016

“…An analytical platform consisted of immunoprecipitation combined with stable isotope labeling‐multiple reaction monitoring MS (SIL/MRM‐MS) assay was recently introduced to detect prostate specific antigen (PSA) isoforms, which are serum markers for prostate cancer (PCa) . The commercially available anti‐PSA was used to pull down all PSA isoforms from complex serum samples.…”