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

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.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:92

Enthalten in:

Analytical chemistry - 92(2020), 12 vom: 16. Juni, Seite 8228-8234

Sprache:

Englisch

Beteiligte Personen:

Frost, Dustin C [VerfasserIn]
Feng, Yu [VerfasserIn]
Li, Lingjun [VerfasserIn]

Links:

Volltext

Themen:

GMW67QNF9C
Journal Article
Leucine
Neoplasm Proteins
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Anmerkungen:

Date Completed 12.02.2021

Date Revised 12.02.2021

published: Print-Electronic

Citation Status MEDLINE

doi:

10.1021/acs.analchem.0c00473

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM309844770