Immunoaffinity Nanoprobe-Based MALDI-TOF MS for Detection of Fragments of N-Telopeptides of Type I Collagen

Abstract

Osteoporosis is one of the major bone-related diseases. Among the biomarkers of bone resorption, type I collagen cross-linked N-telopeptides (NTx) are terminal metabolites specifically derived from the degradation of bone collagen, thus, the level of NTx in urine has been regarded as a highly specific index for bone resorption. Our previous studies have identified a synthetic peptide fragment in the N-terminal of NTx (peptide P2) with highest affinity for anti-NTx antibodies by using enzyme-linked immunosorbent assay and surface plasma resonance-based methods. The objective of this study is to prepare the mouse anti-P2 polyclonal antibodies (anti-P2 Ab) and develop an immunonanoprobe-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for detection of fragment of NTx of type I collagen. Anti-P2 Ab were prepared, purified, characterized, and used to produce Ab-conjugated magnetic nanoparticles (Ab@MNPs) for specifically isolation of different concentrations of Ab-bound P2 standards. The profile of P2 standards which were bound to Ab–NPs was analyzed by combining immunoaffinity MNPs with MALDI-TOF MS. We demonstrated that an immunoaffinity nanoprobe-based MALDI-TOF MS method for detecting the fragment of NTx, and might provide a tool to discover a promising biomarker of osteoporosis. The potential of this method for early diagnosis of osteoporosis will be further investigated by recruiting osteoporosis patients from our collaborative hospitals.