Immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM) is emerging as a potential replacement for enzyme-linked immunosorbent assays (ELISAs). Although ELISAs are often the method of choice to detect protein biomarker candidates, this method is limited by the expense of testing, as well as by the availability of high-quality antibodies for biomarker candidates. Multiple reaction monitoring (MRM) has the advantage of analyzing multiple proteins all at once through multiplexed assays. Interested in how well IP-MRM compared to ELISA, the Liebler research group at the University of Vanderbilt recently published a study that compared the effectiveness of these two methods.1
The researchers used six colon cancer biomarker candidates found in plasma for this comparison; they obtained samples from colon cancer patients and matched healthy controls. The colon cancer biomarkers included metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9). These biomarker candidates can be detected using commercially available ELISAs. The same antibodies used in ELISAs were also available for use in IP-MRM assays, which made it possible to directly compare ElISA and IP-MRM.
For the IP-MRM assays, aldehyde beads (Thermo Scientific) immobilized the relevant antibodies. Next, the research team purified the proteins and performed an MRM analysis on a TSQ Vantage triple-stage quadrupole mass spectrometer (Thermo Scientific).
The researchers analyzed TIMP1 in a single reaction, while they analyzed the other five proteins in a multiplexed reaction. They performed their initial experiments using a “mock plasma” matrix, developing the matrix by spiking proteins into 60 mg/mL of BSA in PBS to produce concentrations ranging from 640 ng/mL to 10 ng/mL. This process found that the limit of detection (LOD) was 2.5 ng/mL for TIMP1.
Next, the researchers performed IP-MRM to detect TIMP1 in the colon cancer and healthy control samples using 50 uL plasma aliquots from 12 individuals. This analysis found that TIMP1 concentrations in plasma were 212 ng/mL and 141 ng/mL for cancer patients and controls, respectively. These findings between cancer patients and healthy individuals were signiﬁcantly diﬀerent (unpaired t-test, p = 0.0028). Interestingly, the ELISA analysis for TIMP1 measured concentrations of 120 ng/mL and 97 ng/ mL for cancer patients and controls, respectively, which were not signiﬁcantly diﬀerent (unpaired t-test, p = 0.06).
Using an identical setup for the mixed proteins, the investigators identified the LOD as between 2 ng/mL and 10 ng/mL. The mean concentration of these ﬁve proteins (determined by IP-MRM) ranged from 8.3 ng/mL (ENG) to 221 ng/mL (COMP). The mean concentrations determined by ELISA in plasma samples from normal and cancer patients ranged from 3.9 ng/mL (ENG) to 208 ng/mL (COMP). The measured values between healthy and cancer plasma samples with either ELISA or IP-MRM yielded only two statistically significant results. A comparison of the average diﬀerence in plasma concentrations measured by IP-MRM versus ELISA for individual candidates indicated that four diﬀerences were signiﬁcant (p < 0.05, paired t-test): TIMP1 (67.1 ng/mL), THBS2 (−13.4 ng/mL), MSLN (−11.7 ng/mL) and ENG (5.2 ng/mL), although no significant differences existed for COMP (−6.4 ng/mL) and MMP9 (13.5 ng/mL).
The overall results indicate that IP-MRM and ELISA give comparable results. The researchers report that both IP-MRM and ELISA measurements were well correlated, with coefﬁcients for all of the analytes except ENG = −.017. They reported that the poor correlation of ENG was likely due to the limited range of ELISA measurements, which was near the LOD of the assay at that particular dilution. All in all, the researchers suggest that IP-MRM analysis may dramatically expand the scope of targeting protein quantification by providing a faster, less costly approach to detect target proteins.
1. Lin, De, et al. (2013) ”Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma,” Journal of Proteome Research, 12(12) (pp. 5996–6003).
Post Author: Emily Humphreys. As a biology undergraduate at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.
While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
And has been a regular contributor to Accelerating Science since 2012.