New Immobilization Chemistry for Probe Affinity Mass Spectrometry

Abstract

Probe affinity mass spectrometry (PAMS) is a technique that combines affinity separations directly with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this approach, a binding molecule, such as an antibody, lectin or receptor, is covalently attached to the surface of a MALDI probe. This permits the analyte of interest to be selectively captured and concentrated on the probe surface prior to MALDI-MS analysis. A major limitation of our initial PAMS immobilization chemistry was that it produced only a relatively small number of binding sites on the probe, as it was based on forming a single monolayer of the binding molecule. Because of this limitation, we have investigated new immobilization chemistries for PAMS that are not confined by monolayer formation and thus allow a larger number of analyte molecules to be captured by the probe. We have developed a new PAMS chemistry that first attaches very high molecular weight (approximately 500,000) dextrans to the MALDI probe, followed by immobilization of the binding molecules to the probe-bound dextrans. Because the size of each dextran molecule is significantly larger than the binding molecule, multiple binding molecules can be linked to the same dextran chain. We have demonstrated that these surfaces possess approximately 500 times more analyte binding sites than probes prepared with our original PAMS chemistry. This chemistry is applicable to any binding molecule that contains a primary amine and is suitable, therefore, for a wide range of applications.

Immune-enrichment of insulin in bio-fluids on gold-nanoparticle decorated target plate and in situ detection by MALDI MS

“…The insulin peaks could be resolved from the background (≥3 times of baseline intensity) even when the insulin level was as low as 0.8 nM. The limit of detection (LOD) at 0.8 nM obtained in standard solution is much lower than the previous reported similar works, in which the tested peptide levels were usually at the range of micromole [25] or several hundred nano-moles [24]. A good linear correlation (R 2  = 0.994) of MS intensity with insulin level was obtained in the range of 0.8–48 nM (Fig.…”

“…A small number of studies have reported the use of this strategy to detect insulin [23–25]. In these reported methods, the antibody of target analyte was immobilized on 2-dimension planar substrate by using complicated chemical reagents [23, 24] or non-specific adsorption [25]. However, the surface area of 2-dimension planar substrate was restricted, limiting the number of immobilized antibodies and the approach of target analytes [23–25].…”

“…However, the surface area of 2-dimension planar substrate was restricted, limiting the number of immobilized antibodies and the approach of target analytes [23–25]. Meanwhile, the complex organic chemicals such as dextran [24] may also introduce more non-target signals to MS analysis. Thus, the sensitivities of these strategies were generally in micromole range, hindering a more wide-spread application for the analysis of real biological fluids [23, 25].…”

DNA methyltransferase expression in the mouse germ line during periods of de novo methylation

“…In probe affinity mass spectrometry (PAMS), a binding molecule (e.g., antibody, lectin, or receptor) is covalently bound to the surface of the MALDI probe allowing for selective capture and concentration on the probe surface before mass spectrometric analysis (Brockman & Orlando, 1995). Different methods of surface functionalization (Brockman & Orlando, 1996), including photoimmobilization (Janecki, Broshears, & Reilly, 2004), can now be used to attach proteins onto surfaces. Wang, Tseng, and Lebrilla (1999) introduced a general concept for producing bioaffinity MALDI probes that could be useful in analyte preconcentration, extraction or purification.…”

Peptide/Protein Separation with Cationic Polymer Brush Nanosponges for MALDI-MS Analysis

“…These on-MALDI-target sample fractionation approaches typically involve direct modification of the target surface to incorporate a selective affinity capture motif, based on either bioselectivity or chemical selectivity. 4,5,6 One limitation of these on-MALDI-target fractionation approaches, is the inherent low loading capacities of the modified target surfaces, which may be limited to as little as a monolayer of captured analyte, or even lower. In an effort to address this limitation, while retaining the advantages of the on-MALDI-target approach, our research has focused on the development of functional brush-polymer modified MALDI targets.…”