21-plex DiLeu Isobaric Tags for High-Throughput Quantitative Proteomics

Anal Chem. 2020 Jun 16;92(12):8228-8234. doi: 10.1021/acs.analchem.0c00473. Epub 2020 May 28.

Abstract

Isobaric tags enable multiplexed quantitative analysis of many biological samples in a single LC-MS/MS experiment. As a cost-effective alternative to expensive commercial isobaric tagging reagents, we developed our own custom N,N-dimethylleucine "DiLeu" isobaric tags for quantitative proteomics. Here, we present a new generation of DiLeu tags that achieves 21-plex quantification in high-resolution HCD MS/MS spectra via distinct reporter ions that differ in mass from each other by a minimum of 3 mDa. The 21-plex set retains the compact tag structure and existing isotopologues of the 12-plex set but includes nine new reporter variants formulated with unique configurations of 13C, 15N, and 2H stable isotopes, each synthesized in-house via a stepwise N-monomethylation synthesis strategy using readily available reagents. Thus, multiplexing capacity is expanded significantly, while preserving the performance and low cost of the previous implementation. We show that 21-plex DiLeu tags generate strong reporter ions following HCD fragmentation of labeled peptides acquired on Orbitrap platforms at a minimum of 60,000 resolving power (at 400 m/z), and we demonstrate accurate 21-plex quantification of labeled K562 human cell line protein digests via single-shot nanoLC-MS/MS analysis on a Q Exactive HF system.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid
  • High-Throughput Screening Assays
  • Humans
  • K562 Cells
  • Leucine / analogs & derivatives
  • Leucine / chemical synthesis
  • Leucine / chemistry*
  • Molecular Structure
  • Neoplasm Proteins / analysis*
  • Proteomics*
  • Tandem Mass Spectrometry

Substances

  • Neoplasm Proteins
  • Leucine