Isobaric labeling techniques are widely used in mass spectrometry-based quantitative proteomics to enable the simultaneous analysis of multiple samples. However, commercial isobaric tags are expensive due to complex synthesis and costly reagents, limiting their use in large-scale studies. Here, we introduce a novel, cost-effective diethylalanine-based isobaric reagent (DeAla), synthesized using diethylated alanine and β-alanine with N-hydroxysuccinimide. The DeAla tag offers several advantages, including improved peptide fragmentation, enhanced protein identification, and competitive pricing. We optimized labeling efficiency and collision energy parameters, demonstrating that DeAla-labeled peptides produce more backbone fragmentation ions and higher XCorr values compared to peptides labeled with N,N-dimethyl leucine (DiLeu) tags. By selectively incorporating stable isotopes, we expanded the multiplexing capacity to 13-plex without increasing structural complexity, achieving baseline resolution in Orbitrap MS/MS acquisition at 60k resolution. Comparative proteomic analyses of two cancer cell lines demonstrated that DeAla labeling outperformed DiLeu tags and showed comparable performance to label-free approaches in terms of protein and peptide identification. Additionally, DeAla provided accurate and reproducible quantification across a dynamic range with minimal technical variability. Overall, the 13-plex DeAla reagents are cost-effective, high-performance isobaric tagging tools that enhance peptide fragmentation and protein identification while ensuring high quantification accuracy, making them valuable for complex quantitative proteomic analyses.
Liu et al. (Mon,) studied this question.