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

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-dimethyl leucine '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 13 C, 15 N, and 2 H 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.

Integrated Label-Free and 10-Plex DiLeu Isobaric Tag Quantitative Methods for Profiling Changes in the Mouse Hypothalamic Neuropeptidome and Proteome: Assessment of the Impact of the Gut Microbiome

“…DiLeu isobaric tags enable up to 21plex quantification during high-resolution MS/MS acquisition. 28 Our DiLeu reagents provided good coefficients of variation (CVs) of 7.9% for 1:1 ratio samples and 11.5% for 16:1 ratio samples and showed a good reproducibility and variance of the protein quantitative ratios between 16:1 replicates. 26 12-Plex DiLeu tags have been employed to develop a highly multiplexed quantitative proteomic and phosphoproteomic method to globally assess protein expression and phosphorylation changes in smooth muscle cells.…”

“…29 Compared with current commercial multiplexed isobaric labels, DiLeu isobaric labeling reagents are attractive due to their cost-effectiveness, comparable performance, high proteome coverage, quantification efficiency, and high multiplexing capability. 26,28,30 In this study, neuropeptides and proteins were extracted from the same mouse brain hypothalamus region, and the relative quantitative changes of neuropeptides and proteins between different groups were measured and compared based on either the precursor ion intensity from MS spectra or DiLeu isobaric tag reporter ion intensities from MS/MS spectra. On one hand, this integrated label-free and 10-plex DiLeu isobaric tag quantitative method enabled the profiling of dynamic changes of neuropeptides and proteins simultaneously in the hypothalamus of mice with distinct gut microbiota environments; on the other hand, the possible connections and interplay between neuropeptides and proteins could be established with this combined approach.…”

Advances in quantitative high‐throughput phosphoproteomics with sample multiplexing

“…This multiplexing limit can ostensibly be expanded upon as multiplexing of up to 21 samples has been reported for the DiLeu chemistries [43].…”

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity

“…Isobaric labeling reagents, such as tandem mass tags (TMT), isobaric tags for relative and absolute quantitation (iTRAQ), isotope-encoded dimethylated leucine or isobaric N, N-dimethyl leucine (DiLeu) and isobaric tag (IBT), permit global glycopeptide quantification with high sample multiplexity (up to 18 parallel samples for TMT and 21 for DiLeu) [13,[74][75][76]. These reagents commonly consist of an amine-reactive group (reacting with the glycopeptide's N-terminus and with the side chain of lysine), a mass balancer and a reporter.…”

“…Multiplexed isobaric labeling reduces the overall LC-MS measurement time and variations introduced between measurements [74,75]. In addition, by pooling all samples together, it boosts the signal of low-abundance species that are otherwise not detectable in any individual sample.…”