Potential for Using Isotopically Altered Metalloproteins in Species-Specific Isotope Dilution Analysis of Proteins by HPLC Coupled to Inductively Coupled Plasma Mass Spectrometry

Abstract

The production and evaluation of an isotopically enriched metalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacterium Acido-thiobacillus ferrooxidans, it was possible to demonstrate the analytical conditions that could be used for the measurement of metalloproteins by on-line IDMS analysis. Rc was chosen because it is a well-characterized protein with an established amino acid sequence and can be produced in suitable quantities using a bacterial recombinant system. Three different forms of the protein were studied by organic and inorganic mass spectrometry: the native form of the protein containing a natural isotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cu isotope, and the nonmetalated apo form. Incorporation of the copper isotopes into the apo form of the protein was determined using a UV-vis spectrophotometric assay and shown to be complete for each of the copper-containing species. The experimental conditions required to maintain the conformational form of the protein with a nonexchangeable copper center were established using +ve electrospray mass spectrometry. A pH 7.0 buffer was found to afford the most appropriate conditions, and this was then used with HPLC-ICP-MS to verify the stability of the copper center by analysis of mixtures of different isotopic solutions. No exchange of the enriched copper isotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pH environment, indicating that species-specific ID-MS analysis of metalloproteins is possible.

Evaluation of gel electrophoresis conditions for the separation of metal‐tagged proteins with subsequent laser ablation ICP‐MS detection

“…Generally it can be said that all results achieved need to be considered very carefully in order to judge whether they are reliable or whether the signals derived from GE-LA-ICP-MS analysis are from contaminants or artefacts. A fully quantitative approach would benefit from the use of isotopically labelled metalloproteins [40,41]. When spiked and equilibrated with the sample, the isotopically labelled species allows metal loss during sample preparation and separation to be compensated for.…”

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

“…The production of the isotopically enriched metalloproteins is usually carried out under in vitro conditions, via incubation of the apo protein (metal-free) with a spike solution, which contains the isotopically enriched metal. [123][124][125] Recently the in vivo synthesis of isotopically enriched metal proteins such as 57 Feferritin 126,127 or 65 Cu-plastocyanin 128 has also been successfully demonstrated, just to give a few examples. Despite the inherent advantage of such speciesspecific standards it is important to ensure that during the demetallation process, which is necessary for the in vitro generation of the apo protein, no degradation of the protein occurs.…”

Metrological Approach for the Quantification of Metalloproteins (EMRP Project HLT05)

“…Thus the spike accounts for any loss or changes in the concentration or conformation of the protein. In contrast to IDMS approaches with organic MS where the labelled protein is produced using either 13 C or 15 N labelled amino acids [7,8], the metal contained within these proteins is used to produce the spike. For this purpose the metal with natural abundance is removed from the protein and replaced by the metal enriched in one isotope.…”

“…The preferable abundance ratio of reference isotope (usually the naturally most abundant isotope) and spike isotope (the one enriched in the spike material) is 1. Should any transformation or losses occur, it can be assumed that the spike is affected in the same way as the analyte; thus the isotope ratio in the analyte will not be changed [13]. When using the metal for the quantification, the sample preparation usually includes a separation step to ensure that the protein under investigation is separated from all other molecules containing the same metal.…”

“…As an example, haemoglobin will be investigated using ID-ICP-MS as well as organic IDMS via characteristic 13 C labelled peptides and the alkaline haematin detergent (AHD) method [16]. The latter was developed by Heuck at al.…”