Dual labeling of biomolecules using MeCAT and DOTA derivatives: application to quantitative proteomics

Abstract

In this study, single and dual labeling of primary amino and thiol groups of target peptides is presented as a proof of concept. The proposed method allows flexible, independent and sequential labeling of the mentioned residues using lanthanides introduced via DOTA-complexes (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The efficiency of the method was optimized using cysteine-containing standard peptides and then applied to bovine serum albumin (BSA) and human serum albumin (HSA) to demonstrate qualitative and quantitative aspects of this strategy. For amino labeling, cysteinyl peptides were immobilized on Sepharose-6B resin and labeled with DOTA-NHS ester followed by metallation with lanthanides. Thiol labeling was carried out using lanthanide-containing metal-coded affinity tags (MeCAT) after elution of peptides from the resin. Complete dual labeling of the standard peptides was demonstrated by liquid chromatography electrospray ionization mass spectrometry, whereas more than 80% of the detected peptides of BSA and HSA were completely dual-labeled. Parallel detection by LC coupled to inductively coupled plasma mass spectrometry (ICP-MS) delivered reliable quantitative information. Thus, the versatile lanthanide choice in both labeling steps allowed estimating primary amino and thiol stoichiometries for the studied samples using different lanthanides. On the other hand, enhancement of ICP-MS signal was achieved as expected when all positions were labeled with the same lanthanide. Finally, linear calibrations of the signal for most of the labeled peptides by standard additions of digested BSA showed a suitable behaviour for quantitative applications and demonstrated the pre-concentration capability of the employed resin.

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA‐based labels and N‐termini DOTA‐labeled peptides

“…However, the application of HCD@HE yielded metal core reporter ions. The ratio amino:thiol function was calculated based on the abundance of the corresponding lanthanide ions from peptides labeled at both the amino and thiol residues using specific DOTA reagents loaded with different lanthanides (NCE > 250%) …”

“…Real‐life samples have been quantified following different analytical strategies including liquid chromatography and gel electrophoresis separations . Similarly, DOTA‐SCN and DOTA‐NHS complexes have been employed to target primary amino groups and have been successfully used in combination with thiol reactive labels to achieve multi‐residual labeling …”

Inductively Coupled Plasma Mass Spectrometry

“…Dual tagging of a protein via its thiol and amino-groups is also possible. 384 More details about the history of immunoassays and detection by ICP-MS are given in a recent review. 385 The detection of electrophoretically separated and electroblotted cytochromes P450 by element-tagged monoclonal antibodies via laser-ablation ICP-SFMS (Element2) was described by Roos et al 386 Cytochromes P450 (CYP) are iron-containing enzymes that play an important role in the metabolism of toxicants and drugs.…”

Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments

“…To enhance the detection sensitivity of this approach, consecutive labelling of different amino acid residues has been also achieved [7]. The analytical robustness of this approach and its suitability for quantitative proteomics utilizing MS/MS analysis has been demonstrated [1,4,[8][9][10][11][12].…”