Protein labelling with mercury tags: fundamental studies on ovalbumin derivatised with p-hydroxymercuribenzoic acid (pHMB)

Abstract

Protein labelling in combination with mass spectrometry is appointed as a modern approach for quantifying biopolymers, especially proteins. With respect to elemental mass spectrometry, specifically inductively coupled plasma-mass spectrometry (ICP-MS), protein labelling approaches are still scarce, although they offer many advantages, e.g. in terms of detection sensitivity. In this fundamental work, we present results on the labelling of ovalbumin with p-hydroxymercuribenzoic acid (pHMB). After optimising the derivatisation procedure, the characterisation of the labelled species is necessary, and thus, the use of molecular MS techniques like MALDI-, and ESI-MS is required. Finally, the detection capabilities of ICP-MS are evaluated on the labelled species. Important factors to consider are the reaction yield, the selectivity, and the stoichiometry of the bioconjugate. For instance, the stoichiometry of the bioconjugate is determined by comparative measurements using MALDI-, and ESI-MS. It can be demonstrated that the label/protein ratio is determined to be $3 : 1 by MALDI-MS, which is lower than the number of expected binding sites (ovalbumin has four free sulfhydryl groups from cysteines). In contrast to these findings, the use of ESI-Q-ToF-MS with its superior mass resolution indicates a stoichiometry of 4 : 1. However, the overall strategy given here on the example of ovalbumin labelling with pHMB might be a promising approach for protein quantification as it provides a significant improvement in terms of detection limits (1 fmol for ovalbumin) in comparison to the use of sulfur as naturally occurring elemental tag.

Evaluation of Nonferrous Metals as Potential In Vivo Tracers of Transferrin-Based Therapeutics

“…An alternative approach to detection and quantitation of exogenous proteins in complex biological matrices relies on placing metal tags on proteins followed by their detection with inductively coupled plasma (ICP) MS [27]. ICP MS is a sensitive technique for measuring metal- and metalloid-containing compounds [28], and in the absence of interferences from ubiquitous metals and plasma-related ions it can allow the limits of detection to be achieved at low fmol level [29, 30].…”

Determination of Thiols in Yeast by HPLC Coupled with LTQ-Orbitrap Mass Spectrometry after Derivatization with p-(Hydroxymercuri)benzoate

“…Based on this characteristic, PHMB has been served as a mass spectrometry tag for counting the number of sulfhydryl groups and disulfide bonds in peptides and proteins (14). PHMB has also been applied to the protein quantification, and it i mproves the detection sensitivity of ovalbumin significantly in comparison to the use of sulfur as naturally occurring elemental tag (15).…”

Quantification of phytochelatins and their metal(loid) complexes: critical assessment of current analytical methodology

“…8), based upon HPLC-ICP-MS measurements monitoring mercury at m/z 202 [47]. Ovalbumin was selected as the protein to study, as it contains 6 cysteine and 16 methionine residues within its structure, giving rise to 22 possible sites for reaction with 22.…”