Chemical tagging with stable isotopes is one of the best established methods for the quantification of proteins using mass spectrometry, especially in non-proliferating cells and tissue. The absolute quantification of proteins is still a challenge. Metal-coded affinity tagging (MeCAT), used to label proteins and peptides with lanthanide ions, allows both, relative and absolute, quantitative determination. MeCAT loaded with lanthanide ions allows the use of inductively coupled plasma mass spectrometry (ICP-MS) enabling very accurate and sensitive quantification of peptides and proteins based on the metal ion signal. Furthermore, multiplex assays are possible that are not limited to 4- or 8-plex analyses when using different lanthanides. Naturally, different lanthanides also lead to different molecular masses for the same labelled peptides which can be distinguished easily. This enables the relative quantification in electrospray MS based on the relative signal intensities of the differentially labelled peptides. We have studied MeCAT labelled peptides, using LC/ESI-MS and LC/ESI-MS/MS with infrared multiphoton dissociation (IRMPD) to show that both the molecular masses and the specific fragments resulting from the MS/MS experiments can be used for relative quantification. The results are compared with high performance liquid chromatography (HPLC)/ICP-MS and direct ICP-MS analysis as standard methods. We show that the ESI and IRMPD based methods deliver quantitative results comparable to ICP-MS.

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.

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