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Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: a review.

Kretschy D, Koellensperger G, Hann S - Anal. Chim. Acta (2012)

Bottom Line: Fundamental methodology of elemental labelling will be highlighted and analytical, as well as biomedical applications will be presented.A special focus will lie on established applications underlining benefits and bottlenecks of such approaches for the implementation in real life analysis.Key research made in this field will be summarized and a perspective for future developments including sophisticated and innovative applications will given.

View Article: PubMed Central - PubMed

Affiliation: University of Natural Resources and Life Sciences, BOKU Vienna, Department of Chemistry, Division of Analytical Chemistry, Muthgasse 18, A-1190 Vienna, Austria.

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LC–ESI-FTICR-MS (A) and LC–ICP-MS (B) chromatograms of standard peptides p1 and p2 dually labelled in the amino and thiol with DOTA-NHS-Ho and MeCAT-IA-Tm, respectively. The extracted ion chromatograms (EIC) of the dual-labelled p1 and p2 are also presented in (A). The labelling positions are indicated by an asterisk (amino positions) or dot (thiol positions) in the sequence.
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fig0020: LC–ESI-FTICR-MS (A) and LC–ICP-MS (B) chromatograms of standard peptides p1 and p2 dually labelled in the amino and thiol with DOTA-NHS-Ho and MeCAT-IA-Tm, respectively. The extracted ion chromatograms (EIC) of the dual-labelled p1 and p2 are also presented in (A). The labelling positions are indicated by an asterisk (amino positions) or dot (thiol positions) in the sequence.

Mentions: Esteban-Fernandez et al. [33] addressed absolute quantification of lysozyme and BSA on the peptide level using RP-ICP-MS (Polaris 3 C18-Ether, 150 mm × 1 mm, 5 μm; Varian), while complementary RP-ESI-MS of the digested and labelled proteins served as identification technique. For RP-ESI-MS 6 different synthetic standard peptides labelled with Eu-MeCAT-maleimide were employed as standards and were selected because of their different behaviours in reversed phase separations and good identification capabilities. For quantification of the Eu-MeCAT-maleimide labelled peptides resulting from BSA and lysozyme digestion via RP-ICP-MS species unspecific isotope dilution analysis (IDA) with post-column addition of a Eu2O3 spike was performed. Summarizing, this study was the first successful application of the MeCAT technique for cysteine labelling applied for absolute protein quantification and can serve as an example for the fruitful combination of organic and inorganic MS together with elemental labelling. Recently, a proof of concept study for dual labelling of thiol and amine groups of peptides via MeCAT-iodoacetamide and DOTA-NHS-ester combined with lanthanides was published by this group [66]. The method was optimized for cysteine containing standard peptides and applied for BSA and HSA, offering capabilities for tracking peptide/protein reactions or estimation of thiol and primary amine groups. In principle, the peptides were first immobilized on Sepharose 6B resins and labelled with the DOTA-NHS ester followed by coordination of lanthanides to the complex. Afterwards, the complexes were eluted and cysteine residues were labelled with a lanthanide loaded MeCAT-iodoacetamide. Detection was maintained parallel by the use of RP-ESI-MS (applying a Zorbax 300 SB C18 column) for determination of labelling completeness and yield, and RP-ICP-MS utilizing the same column for gathering of relative quantitative information (Fig. 3). A drawback of this application was the lack of accuracy of the quantitative determinations, which was suggested to be due to systematic errors.


Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: a review.

Kretschy D, Koellensperger G, Hann S - Anal. Chim. Acta (2012)

LC–ESI-FTICR-MS (A) and LC–ICP-MS (B) chromatograms of standard peptides p1 and p2 dually labelled in the amino and thiol with DOTA-NHS-Ho and MeCAT-IA-Tm, respectively. The extracted ion chromatograms (EIC) of the dual-labelled p1 and p2 are also presented in (A). The labelling positions are indicated by an asterisk (amino positions) or dot (thiol positions) in the sequence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475989&req=5

fig0020: LC–ESI-FTICR-MS (A) and LC–ICP-MS (B) chromatograms of standard peptides p1 and p2 dually labelled in the amino and thiol with DOTA-NHS-Ho and MeCAT-IA-Tm, respectively. The extracted ion chromatograms (EIC) of the dual-labelled p1 and p2 are also presented in (A). The labelling positions are indicated by an asterisk (amino positions) or dot (thiol positions) in the sequence.
Mentions: Esteban-Fernandez et al. [33] addressed absolute quantification of lysozyme and BSA on the peptide level using RP-ICP-MS (Polaris 3 C18-Ether, 150 mm × 1 mm, 5 μm; Varian), while complementary RP-ESI-MS of the digested and labelled proteins served as identification technique. For RP-ESI-MS 6 different synthetic standard peptides labelled with Eu-MeCAT-maleimide were employed as standards and were selected because of their different behaviours in reversed phase separations and good identification capabilities. For quantification of the Eu-MeCAT-maleimide labelled peptides resulting from BSA and lysozyme digestion via RP-ICP-MS species unspecific isotope dilution analysis (IDA) with post-column addition of a Eu2O3 spike was performed. Summarizing, this study was the first successful application of the MeCAT technique for cysteine labelling applied for absolute protein quantification and can serve as an example for the fruitful combination of organic and inorganic MS together with elemental labelling. Recently, a proof of concept study for dual labelling of thiol and amine groups of peptides via MeCAT-iodoacetamide and DOTA-NHS-ester combined with lanthanides was published by this group [66]. The method was optimized for cysteine containing standard peptides and applied for BSA and HSA, offering capabilities for tracking peptide/protein reactions or estimation of thiol and primary amine groups. In principle, the peptides were first immobilized on Sepharose 6B resins and labelled with the DOTA-NHS ester followed by coordination of lanthanides to the complex. Afterwards, the complexes were eluted and cysteine residues were labelled with a lanthanide loaded MeCAT-iodoacetamide. Detection was maintained parallel by the use of RP-ESI-MS (applying a Zorbax 300 SB C18 column) for determination of labelling completeness and yield, and RP-ICP-MS utilizing the same column for gathering of relative quantitative information (Fig. 3). A drawback of this application was the lack of accuracy of the quantitative determinations, which was suggested to be due to systematic errors.

Bottom Line: Fundamental methodology of elemental labelling will be highlighted and analytical, as well as biomedical applications will be presented.A special focus will lie on established applications underlining benefits and bottlenecks of such approaches for the implementation in real life analysis.Key research made in this field will be summarized and a perspective for future developments including sophisticated and innovative applications will given.

View Article: PubMed Central - PubMed

Affiliation: University of Natural Resources and Life Sciences, BOKU Vienna, Department of Chemistry, Division of Analytical Chemistry, Muthgasse 18, A-1190 Vienna, Austria.

Show MeSH