Opt-TMT: An optimized and scaled-down TMT labeling strategy for limited sample

Mass spectrometry-based proteomics has evolved and currently requires minimal sample quantities. However, manufacturers' isobaric labeling protocols, such as Tandem Mass Tag (TMT), are still designed for larger sample amounts, leading to significant costs and limiting research possibilities. Here, we present an optimized isobaric labeling protocol (Opt-TMT) that maintains high labeling efficiency while substantially reducing reagent consumption and sample requirements. We achieved consistent labeling efficiency even with peptide quantities as low as 6-15 μg per channel by adjusting reaction conditions, including volume reduction and increased peptide concentration. Importantly, our protocol reduces reagent costs by up to 90 % compared to the commercial protocol, while maintaining labeling efficiency above 99 %. This cost-effective approach addresses key challenges in proteomic research, especially for studies involving limited biological material or post-translational modification analyses. The Opt-TMT method provides a practical solution for researchers seeking to maximize the utility of isobaric labeling while minimizing resource expenditure, without compromising analytical quality. SIGNIFICANCE: The Opt-TMT protocol provides a major advancement for proteomics by making isobaric labeling both cost-effective and scalable to very limited sample amounts. By reducing reagent costs up to 90 % without compromising labeling efficiency (>99 %), this approach enables high-quality quantitative proteomics for studies where material is scarce, such as patient-derived samples, biopsies, or post-translational modification analyses. Importantly, Opt-TMT broadens access to TMT-based workflows for laboratories with limited resources, while preserving analytical robustness. This work contributes a practical and impactful methodological improvement that directly benefits both fundamental and applied proteomics research.