Unmasking low‐abundance peptides from human blood plasma and serum samples by a simple and robust two‐step precipitation/immunoaffinity enrichment method

Abstract

Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.

Solid-Phase Extraction Strategies to Surmount Body Fluid Sample Complexity in High-Throughput Mass Spectrometry-Based Proteomics

“…In the case of enrichment strategies, in general more information about the analyte of investigation is already available and the research question has focused on a very specific group of proteins or even a single protein. An elegant example of immunoaffinity (IA) enrichment was shown by Halfinger and coworkers [ 61 ], which involved a thermal protein precipitation step, followed by liquid-liquid precipitation before IA chromatography to quantify the low-abundance heart failure marker N-terminal pro-B-type natriuretic peptide (NT-proBNP). This example showed a combined preparation method to deplete high-abundant proteins followed by concentration of a specific low-abundant protein through immunoaffinity.…”

Application and Challenges in Using LC–MS Assays for Absolute Quantitative Analysis of Therapeutic Proteins in Drug Discovery

“…IC sample cleanup is well suited for nanoLC and Review | Zheng, Mehl, Zhu, Xin & Olah Bioanalysis (2014) 6 (6) had been applied for the quantification of protein and peptide biomarkers in serum [73,[92][93][94].IC sample preparation is compatible with nanoLC for two main reasons: IC removes the bulk of sample matrix, which comprises the majority of the sample mass, the matrix could overload the column capacity, leading to poor chromatography and high background; and IC process can concentrate analytes, thereby lead to low LOQs or increase the assay sensitivity.…”