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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.

Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as (2)D, (13)C, (15)N, or (18)O, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-hydroxymercuribenzoic acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [(199)Hg]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pattern deconvolution was applied to extract the quantitative data from the mass spectra. Good agreement with the expected values was obtained for the relative insulin quantification, and the recovery for insulin applying the absolute quantification workflow was between 90% and 110% with an RSD of better than 5% in the low picomole range.

Since first reports on the impact of metals such as manganese and cobalt on erythropoiesis were published in the late 1920s, cobaltous chloride became a viable though not widespread means for the treatment of anaemic conditions. Today, its use is de facto eliminated from clinical practice; however, its (mis)use in human as well as animal sport as an erythropoiesis-stimulating agent has been discussed frequently. In order to assess possible analytical options and to provide relevant information on the prevalence of cobalt use/misuse among athletes, urinary cobalt concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) from four groups of subjects. The cohorts consisted of (1) a reference population with specimens of 100 non-elite athletes (not being part of the doping control system), (2) a total of 96 doping control samples from endurance sport athletes, (3) elimination study urine samples collected from six individuals having ingested cobaltous chloride (500 µg/day) through dietary supplements, and (4) samples from people supplementing vitamin B12 (cobalamin) at 500 µg/day, accounting for approximately 22 µg of cobalt. The obtained results demonstrated that urinary cobalt concentrations of the reference population as well as the group of elite athletes were within normal ranges (0.1-2.2 ng/mL). A modest but significant difference between these two groups was observed (Wilcoxon rank sum test, p < 0.01) with the athletes' samples presenting slightly higher urinary cobalt levels. The elimination study urine specimens yielded cobalt concentrations between 40 and 318 ng/mL during the first 6 h post-administration, and levels remained elevated (>22 ng/mL) up to 33 h. Oral supplementation of 500 µg of cobalamin did not result in urinary cobalt concentrations > 2 ng/mL. Based on these pilot study data it is concluded that measuring the urinary concentration of cobalt can provide information indicating the use of cobaltous chloride by athletes. Additional studies are however required to elucidate further factors potentially influencing urinary cobalt levels.

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