Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study

Abstract

Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis, and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican, and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.

Figure 1

The comparison of protein quantification using different representative peptides derived from the same protein. A) The plasma concentration of lumican across the 60 plasma samples calculated based on three different representative peptides that were measured by the SRM assay. B) The correlation of lumican concentration in natural log scale using two different representative peptides: SLEYLDLSFNQIAR and SLEDLQLTHNK. C) The lumican plasma concentration profiles categorized in groups (NL, CP and PDAC) based on the quantification of three different representative peptides. The lines represent the mean concentration in each category.

Figure 2

The correlation of replicate plasma concentrations in natural log scale for the four proteins measured in the 60 plasma samples from the pancreatic cancer cohort, the diseased and non-diseased controls.

Figure 3

The comparison of quantification of the SRM assay versus the corresponding ELISA assay in the same plasma samples. A) The tissue inhibitor of metalloproteinase 1 (TIMP-1) plasma concentration profiles categorized in groups (NL, CP and PDAC): SRM vs ELISA. SRM detected a higher concentration of the target protein in a given plasma sample. B) Comparison of fold changes of TIMP1 between the comparison groups (NL, CP and PDAC) using the mean concentration of each group. C) The correlation of SRM measurement for tissue inhibitor of metalloproteinase 1 versus the corresponding ELISA measurement at the individual plasma sample level (natural log scale).

Figure 4

The plasma concentration profiles of the four protein candidates (14-3-3 protein sigma, gelsolin, lumican, and tissue inhibitor of metalloproteinase 1) in each studied groups: NL, CP and PDAC.

Figure 5

The ROC curves of protein 14-3-3 protein sigma, gelsolin, lumican and tissue inhibitor of metalloproteinase 1.

Figure 6

Scatter plot of protein plasma concentration versus the corresponding patient age for 14-3-3 protein sigma, gelsolin, lumican and tissue inhibitor of metalloproteinase 1.