102-Plex Approach for Accurate and Multiplexed Proteome Quantification

Abstract

Hyperplexing approaches have been aimed to meet the demand for large-scale proteomic analyses. Currently, the analysis capacity has expanded to up to 54 samples within a single experiment by utilizing different isotopic and isobaric reagent combinations. In this report, we propose a super multiplexed approach to enable the analysis of up to 102 samples in a single experiment, by the combination of our recently developed TAG-TMTpro and TAG-IBT16 labeling. We systematically investigated the identification and quantification performance of the 102-plex approach using the mixtures of E. coli and HeLa peptides. Our results revealed that all labeling series demonstrated accurate and reliable quantification performance. The combination of TAG-IBT16 and TAG-TMTpro approaches expands the multiplexing capacity to 102 plexes, providing a more multiplexed quantification method for even larger-scale proteomic analysis. Data are available via ProteomeXchange with the identifier PXD042398.

A Tutorial Review of Labeling Methods in Mass Spectrometry-Based Quantitative Proteomics

“…However, these methods limited the protease to LysC to create double amine groups on peptide termini . TAG-TMTpro method was reported as a more universal method. , It introduces mass differences by labeling tert -butyloxycarbonyl (Boc) protected alanine or glycine amino acid residues into peptide amine groups, followed by the deprotection and isobaric labeling on newly introduced amine groups in these amino acid residues. This method allowed for up to 54-plex quantification when combined with 18-plex TMTpro and up to 102-plex when combined with 18-plex TMTpro and 16-plex IBT isobaric tagging. , …”

“…However, these strategies inherit issues from both precursor and reporter ion-based quantification methods, including increased spectral complexity at the precursor level, redundant MS2 spectra, and reporter ion ratio distortion problem . Also, when combining multiple sets of isobaric tagging, missing values of peptide identification may happen between different isobaric groups due to the ionization efficiency variation of isobaric tags, which will influence the reproducibility of proteins identified between groups …”