A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum

Abstract

Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 μg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.

Figure 1

PRISM-SRM workflow for sensitive detection and quantification of AGR2 in urine and serum.

Figure 2

Calibration curve for quantifying AGR2 using diluted urine (20 ng/μL) as the matrix. Recombinant AGR2 with a concentration range from 0.1 to 500 pg/μL was spiked into the diluted urine. The inset plot shows details of the low concentration points. Except for the high concentration point, the error bars of the other concentration points are smaller than the size of the points (Table S1).

Figure 3

Extracted ion chromatograms (XIC) of transitions monitored for AGR2 signature peptides LPQTLSR and HLSPDGQYVPR in a prostate cancer urine sample (A) without PRISM; (B) with PRISM. LPQTLSR: 407.7/351.2 (red), 407.7/476.3 (purple), 407.7/604.3 (blue); HLSPDGQYVPR: 634.8/1018.5 (red), 634.8/1131.6 (blue). Internal standards (top XIC) were spiked at10 fmol/μL. The blue arrows in (A) indicate the locations of expected SRM peak apices of light peptides (bottom XIC) based on the retention time of heavy internal standards.

Figure 4

Correlation between PRISM-SRM and ELISA measurements of AGR2 in urine samples.

Figure 5

AGR2 as a potential urine biomarker for prostate cancer. (A) AGR2 concentrations between non-cancer and cancer subjects (Kolmogorov-Smirnov test, P = 0.068); (B) AGR2/PSA concentration ratios between non-cancer and cancer subjects (Kolmogorov-Smirnov test, P = 0.026).