TY - JOUR

T1 - Quantification of the soluble Receptor of Advanced Glycation End-Products (sRAGE) by LC-MS after enrichment by strong cation exchange (SCX) solid-phase extraction (SPE) at the protein level

AU - Klont, Frank

AU - Joosten, Marc R

AU - Ten Hacken, Nick H T

AU - Horvatovich, Péter

AU - Bischoff, Rainer

N1 - Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2018/12/28

Y1 - 2018/12/28

N2 - The study of low abundant proteins contributes to increasing our knowledge about (patho) physiological processes and may lead to the identification and clinical application of disease markers. However, studying these proteins is challenging as high-abundant proteins complicate their analysis. Antibodies are often used to enrich proteins from biological matrices prior to their analysis, though antibody-free approaches have been described for some proteins as well. Here we report an antibody-free workflow on the basis of strong cation exchange (SCX) enrichment and liquid chromatography-mass spectrometry (LC-MS) for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). sRAGE was quantified in serum at clinically relevant low to sub ng mL(-1) levels. The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and was compared to an antibody-based LC-MS sRAGE method. The SCX-based method builds upon the bipolar charge distribution of sRAGE, which has a highly basic N-terminal part and an acidic C-terminal part resulting in an overall neutral isoelectric point (pI). The highly basic N-terminal part (pI(calculated) = 10.3) allowed for sRAGE to be enriched by SCX at pH 10, a pH at which most serum proteins do not bind. This study shows that ion exchange-based enrichment is a viable approach for the LC-MS analysis of several low abundant proteins following a thorough analysis of their physical-chemical properties. (C) 2018 The Authors. Published by Elsevier B.V.

AB - The study of low abundant proteins contributes to increasing our knowledge about (patho) physiological processes and may lead to the identification and clinical application of disease markers. However, studying these proteins is challenging as high-abundant proteins complicate their analysis. Antibodies are often used to enrich proteins from biological matrices prior to their analysis, though antibody-free approaches have been described for some proteins as well. Here we report an antibody-free workflow on the basis of strong cation exchange (SCX) enrichment and liquid chromatography-mass spectrometry (LC-MS) for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). sRAGE was quantified in serum at clinically relevant low to sub ng mL(-1) levels. The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and was compared to an antibody-based LC-MS sRAGE method. The SCX-based method builds upon the bipolar charge distribution of sRAGE, which has a highly basic N-terminal part and an acidic C-terminal part resulting in an overall neutral isoelectric point (pI). The highly basic N-terminal part (pI(calculated) = 10.3) allowed for sRAGE to be enriched by SCX at pH 10, a pH at which most serum proteins do not bind. This study shows that ion exchange-based enrichment is a viable approach for the LC-MS analysis of several low abundant proteins following a thorough analysis of their physical-chemical properties. (C) 2018 The Authors. Published by Elsevier B.V.

KW - Antibodies, Immobilized/chemistry

KW - Biomarkers/blood

KW - Cation Exchange Resins/chemistry

KW - Chromatography, High Pressure Liquid

KW - Humans

KW - Isoelectric Point

KW - Limit of Detection

KW - Pulmonary Disease, Chronic Obstructive/diagnosis

KW - Receptor for Advanced Glycation End Products/analysis

KW - Solid Phase Extraction/methods

KW - Spectrometry, Mass, Electrospray Ionization

U2 - 10.1016/j.aca.2018.09.050

DO - 10.1016/j.aca.2018.09.050

M3 - Article

C2 - 30392668

VL - 1043

SP - 45

EP - 51

JO - Analytica Chimica Acta

JF - Analytica Chimica Acta

SN - 0003-2670

ER -