Thiol reactive tandem mass tags for quantitative top-down proteomics

Here, we describe the development of a labeling strategy for TDP targeting thiol groups of cysteine residues with iodoTMTsixplex. While this method inherently excludes the quantification of cysteine-free proteoforms, it provides the opportunity of sixplexing and the implementation of multidimensional separation schemes. The approach was tested using both a high/low-pH RP-LC and a gel-eluted liquid fraction entrapment electrophoresis (GelFrEE) x RP-LC separation. Over- and underlabeling rates were determined and MS/MS parameters were optimized to enable parallel protein identifications and quantification. Finally, a complex two proteome interference model was applied to demonstrate the high accuracy of the developed quantification method.