Fundamentals: How Do We Calculate Mass, Error, and Uncertainty in Native Mass Spectrometry?

Mass spectrometry (MS) is uniquely powerful for measuring the mass of intact proteins and other biomolecules. New applications have expanded intact protein analysis into biopharmaceuticals, native MS, and top-down proteomics, all of which have driven the need for more automated data-processing pipelines. However, key metrics in the field are often not precisely defined. For example, there are different views on how to calculate uncertainty from spectra. This Critical Insight will explore the different definitions of mass, error, and uncertainty. It will discuss situations where different definitions may be more suitable and provide recommendations for best practices. Targeting both beginners and experts, the goal of the discussion is to provide a common foundation of terminology, enhance statistical rigor, and improve automation of data analysis.