U.S. Department of Energy

Pacific Northwest National Laboratory

Antibody-Independent, Deep-Dive Targeted Quantification of Proteins at 10 pg/mL Levels in Non-Depleted Human Serum/Plasma

Introduction 

Targeted proteomics approaches, such as selected reaction monitoring (SRM), have emerged as powerful tools for sensitive, quantitative protein analysis in systems biology and biomedical research. Chromatography or affinity based sample pre-fractionation/enrichment is typically performed before SRM analysis when higher sensitivity is needed, especially in the analysis of complex biological samples. However, even with these existing techniques targeted quantitative analysis of extremely low abundance (e.g., <50 pg/mL) proteins in complex biological samples, such as serum or plasma, remains challenging. To address this need, we developed an antibody-independent, two-dimensional (2D) offline liquid chromatography (LC)-based Deep-Dive (DD)-SRM approach for quantification of proteins at low pg/ml levels in human serum/plasma without the need for major protein depletion.

 

Methods 

In DD-SRM, offline 2D-LC separation is used to separate/enrich target peptides before their final LC-SRM analysis. Four milligram of tryptic peptides of non-depleted plasma spiked with heavy standards were first separated on a high-flow low-pH RPLC on a 4.6 mm×150 mm C18 column. The target fractions were further separated on a second-dimension high-pH capillary LC on a 200 μm×75 cm C18 column and target-peptide-containing fractions were selected by on-line LC-SRM monitoring. The target fractions were finally subjected to low-pH LC-SRM analysis using a100 μm×10 cm C18 column. The LC separations were performed using a Waters UPLC and the SRM data were acquired on a ThermoScientific TSQ Vantage triple quadrupole mass spectrometer. Data was analyzed using Skyline software.

 

Preliminary Data 

The DD-SRM concept and approach is based on the following considerations: 1) the orthogonal low-high pH 2D-RPLC separation provides a high-resolution separation that significantly reduces background and potential interference, allowing for highly effective enrichment of target peptides, minimizing sample handling/reconditioning and leading to high recovery; 2) the sequential use of high-flow LC, microLC and nanoLC in the first, second and third dimension separations, respectively, allows for extremely high initial sample loading (e.g., 4 mg of peptides, equivalent to approximately 200 mL of plasma) and increased sensitivity for detecting the target peptides in the enriched fractions with greatly reduced total peptide loading. These advantages should lead to ultra-sensitive targeted quantification of low-abundance proteins in highly complex biological samples.

 

To provide a proof of principle, a calibration curve was built through a spike-in experiment using prostate-specific antigen (PSA) protein and pooled healthy female plasma. PSA was spiked in the female plasma at concentrations of 0, 0.01, 0.025, 0.05, 0.1, 0.5, 1.0 and 10.0 ng/mL, together with 100 fmol heavy peptide standards. The results showed that the DD-SRM method achieved unprecedentedly sensitive detection of PSA with a limit of quantification (LOQ) at 10 pg/mL in non-depleted human plasma, without the need for any immunoaffinity reagents. A linear range of over 4 orders of magnitude in concentration was achieved (R2 = 0.99), and the CV among three repeats was less than 5%. DD-SRM also allowed for confident detection of 4 endogenous proteins IL13, IL8, TNFA, and IL9 with concentrations ranging from 7 to 30 pg/mL in pooled healthy female plasma. These results demonstrated that the DD-SRM approach represents a major advance in the sensitivity of targeted protein quantification and holds great promise in applications such as verification of extremely low-abundance biomarker candidates without available antibodies.

 

Novel aspect 

A new antibody-independent DD-SRM method allows for ultra-sensitive targeted protein quantification (e.g., 10 pg/mL level in non-depleted serum/plasma). 

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