Absolute and Relative Protein Quantification with the Use of Isotopically Labeled p-Hydroxymercuribenzoic Acid and Complementary MALDI-MS and ICPMS Detection

Abstract

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.

Phosphorus and osmium as elemental tags for the determination of global DNA methylation—A novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies

“…Such an approach enables for enhanced quantification of labeled biomolecules, based on exceptional features of elemental mass spectrometry in terms of sensitivity, selectivity, multielemental and isotopic capabilities [36,37]. In the context of this work, the use of osmium tetroxide in nucleic acid research should be highlighted.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…Their availability finally induced a paradigm shift, since it became possible to analyze elements that are highly susceptible to interference, which finally allows the number of elements that are easily detected by ICP-MS to be extended to covalently bound hetero atoms such as phosphorus, sulfur, selenium, or even halogens, which show a widespread distribution in all classes of chemical substances. 24 More recently, labeling approaches that utilize an ICP-MS detectable element have gained a great deal of interest, in particular in the life sciences, as such approaches can be used to allow those molecules that naturally contain no detectable element tag to be detectable by ICP-MS. [24][25][26][27] Meanwhile appropriate reaction chemistry has been developed that allows the direct covalent labeling of a bio molecule with elements such as Hg [28][29][30] or I, 31,32 both of which can be detected with high sensitivity. In terms of flexibility, labeling with lanthanides using bi-functional chelating agents, which are covalently bound to the targeted bio molecule, and which form highly stable complexes with the metal ion, represents the current state of the art within this field.…”

“…More recently such approaches have been used for the successful quantification of proteins such as insulin or ovalbumin, respectively. 28,29 Cartwright et al described the application of tris(2,4,6-trimethoxyphenyl)-phosphonium propylamine bromide (TMPP) to label compounds containing carboxylic acid residues with phosphorus. 198 HPLC hyphenated to high-resolution sector field ICP-MS operated at medium resolution has been used to detect the labeled compounds via their phosphorus content.…”

Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics

“…On the other hand, artificial elemental labeling makes almost all biomolecules ICP-MS-detectable [15]. Thus, quantification of biomolecules using I [16], Fe [17], Pt [18], Hg [19], or lanthanides [20] as tags have been reported. Particularly remarkable is the labeling strategy using bi-functional chelating agents that can harbor a metal and have a second functional group for specific covalent interaction with the target biomolecule.…”