TY - JOUR A1 - Schwarz, G. A1 - Müller, Larissa A1 - Beck, S. A1 - Linscheid, M.W. T1 - DOTA based metal labels for protein quantification: a review N2 - Today, quantitative data play a pivotal role in the understanding of biological processes. This is particularly true for the proteome: protein quantification always follows protein identification. To obtain useful and reliable quantitative data, rather sophisticated strategies using electrospray and MALDI mass spectrometry have been developed, which allow relative and sometimes even absolute quantification. All of those strategies have merits and limitations. In order to overcome some of these limits, methods based on the reliable and sensitive detection and quantification of heavy metals present in proteins using inductively coupled plasma (ICP)-MS have been reported. With specific labels carrying heavy metals, the applicability of ICP-MS has been extended to almost every protein. One of such covalently bound metal tags, allowing the quantification of low abundant proteins, uses 1,4,7,10-tetraazacyclododecane N,N',N'',N''-tetraacetic acid (DOTA) chelate complexes carrying lanthanides as the metal core. In this review the scope and limitations of peptide and protein quantification will be addressed. The metal tags do not only provide low detection limits, but also due to the large number of different lanthanides and lanthanide isotopes, multiplexing capabilities and previously unknown accuracy based on inherently possible isotope dilution methods came into reach. The developed workflows, including electrophoretic and chromatographic separation and preconcentration techniques, will be addressed to allow a comparison with already established procedures. KW - Protein quantification KW - ICP-MS KW - DOTA KW - Labeling PY - 2014 U6 - https://doi.org/10.1039/c3ja50277e SN - 0267-9477 SN - 1364-5544 VL - 29 IS - 2 SP - 221 EP - 233 PB - Royal Society of Chemistry CY - London AN - OPUS4-30730 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Esteban-Fernández, D. A1 - Bierkandt, Frank A1 - Linscheid, M.W. T1 - MeCAT labeling for absolute quantification of intact proteins using labelspecific isotope dilution ICP-MS N2 - Sensitive, accurate and fast absolute quantification of intact proteins is reported using metal coded affinity tags (MeCATs) combined with gel electrophoresis (GE) separation, inductively coupled plasma mass spectrometry (ICP-MS) detection and label-specific isotope dilution analysis quantification. Two different approaches were investigated concerning the introduction of the sample into ICP-MS, mineralization of the gel and direct laser ablation (LA) of the spot. Three major advantages are remarkable in the proposed method. First, not only heteroatom-containing proteins but also all labeled proteins in the sample can be absolutely quantified preparing an isotopically enriched tracer. On the other hand, highly sensitive determination in the amol range can be performed, considerably reducing the analysis time due to the straightforward measurements by ICP-MS. Furthermore, the studied method allows accurate quantifications with different sample-to-spike ratios and using stock spikes stored for months. After the first characterization using standard proteins, human serum albumin (HSA) and transferrin (Tf) were determined in human serum to test the applicability of the method to biological samples. The results show satisfactory quantifications for the studied proteins despite the difference in concentration and the similarity in migration distances. PY - 2012 U6 - https://doi.org/10.1039/c2ja30137g SN - 0267-9477 SN - 1364-5544 VL - 27 IS - 10 SP - 1701 EP - 1708 PB - Royal Society of Chemistry CY - London AN - OPUS4-27777 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schwarz, Gunnar A1 - Beck, S. A1 - Weller, Michael G. A1 - Linscheid, M.W. T1 - MeCAT - new iodoacetamide reagents for metal labeling of proteins and peptides N2 - Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metalcoded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labelling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent. KW - Quantitative proteomics KW - Mass spectrometry KW - Metal labeling KW - Stable isotope labeling KW - Lanthanide DOTA KW - Metall-Markierung KW - Reagenzien KW - Protein-Quantifizierung KW - Lanthaniden KW - Multiplexing PY - 2011 U6 - https://doi.org/10.1007/s00216-011-5189-7 SN - 1618-2642 SN - 1618-2650 VL - 401 IS - 4 SP - 1203 EP - 1209 PB - Springer CY - Berlin AN - OPUS4-23949 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -