NHS-Ester Tandem Labeling in One Pot for 48-Plex Proteomics

ABSTRACT: Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid isotope labeling strate-gies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quanti-tative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample losses, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotop-ic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of bio-logical and clinical proteomics applications.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yu Lu  

LAB HEAD: Yu Lu

PROVIDER: PXD024780 | Pride | 2022-02-17

REPOSITORIES: Pride