13-Plex DeAla Isobaric Reagents for High-Throughput Proteome Quantification.

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.