Targeted Quantification of Low ng/mL Level Proteins in Human Serum without Immunoaffinity Depletion

Abstract

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

“…However, there are additional strategies that enable MRM-based approaches to reach lower abundance proteins. Multidimensional separations coupled with MRM allow the detection of proteins at the low ng/ml to high pg/ml range in human plasma (47,58). For example, leptin has been previously reported at a concentration of Ͻ30 ng/ml in both human plasma and DBS samples using a singleplex immunoassay (44) We were not able to detect leptin in DBS samples using 1D LC/MRM-MS as described in this manuscript.…”

Current Trends in Magnetic Particle Enrichment for Mass Spectrometry-Based Analysis of Cardiovascular Protein Biomarkers

“…Antibodies are the preferred biomolecule for immunocapture due to their high affinity and antigen selectivity. Nevertheless, specific antibodies require long development times and are relatively expensive [81]. Affinity molecules other than antibodies, such as aptamers, may be used for capturing proteins from complex matrices.…”

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential

“…Nonetheless, at the low end of the quantification scale, 31 proteins were found to have concentrations between 452 pg/mL and 10 ng/mL in the control plasma measured. In a separate example, the "PRISM" (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) technique was implemented to quantify select proteins in depleted [85] and undepleted [86] human serum/plasma. The LOQs were found to be at low pg/mL levels with prior depletion (via IgY14) and at mid pg/mL levels without this form of pre-treatment.…”