Metal-Coded Affinity Tag Labeling: A Demonstration of Analytical Robustness and Suitability for Biological Applications

Abstract

Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanide-harboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well-established technique for assessing concentrations down to low attomole ranges. This work investigates the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromatography/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MS(n)). Focus was given toward the separation behavior of labeled peptides and the dynamic range of detection and peptide charge distribution. Furthermore, the stability of MeCAT under harsh analytical conditions was investigated. With the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of changes in an Escherichia coli proteome, we successfully addressed the suitability to utilize MeCAT on biological samples. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Therefore, this technique is suitable to common proteomic workflows and enables relative as well as absolute differential peptide quantification.

Cysteine tagging for MS‐based proteomics

“…After pooling of the samples and affinity enrichment, RP-LC ESI MS is used to identify and relatively quantify; ICP MS allows detection and absolute quantitation. Reprinted with permission from Ahrends et al (2009). retention times for doubly labeled peptides (i.e., containing two cysteines) was observed; however, the average standard deviation for relative quantitation was still less than 15%. The differential use of labels with terbium, holmium, thulium, and lutetium seemed appropriate for relative 4-plex quantitation.…”

Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase

“…Next, the fragment was quickly rinsed with ultrapure water, dried in a SpeedVac, and incubated overnight with 0.6 g of ProA (purified from L. pneumophila supernatant via AEC) in 40 mM Tris-HCl (pH 7.5), 1 M ZnCl 2 . Trypsin proteolysis of ProA-digested PlaC-His and determination of generated PlaC peptides via nano-LC/ESI-MS were carried out according to a published protocol (40). The ProA-specific cleavage sites in PlaC were determined by exact peptide mass analysis and fragmentation of the ionized peptides applying collision-induced fragmentation.…”

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part II. Applications

“…However, CYP2E1 levels appear to be modulated to some extent by the compounds So far, the previous applications have been related to the labelling of antibodies used for immune-reactions, but in the following example it should be demonstrated that this procedure can also be applied for proteins directly, as it was discussed in a review article recently. 370 Ahrends et al 371,372 used metal-coded affinity tags (MeCAT) for direct labelling of standard proteins and eye lens proteins for quantitative ICP-SFMS. As MeCAT reagents they chose derivatives of the lanthanide chelating DOTA to which a cysteine reactive maleimide moiety was attached via a spacer.…”