Due to their oftentimes ambiguous nature, phosphopeptide positional
isomers can present challenges in bottom-up mass spectrometry-based
workflows as search engine scores alone are often not enough to
confidently distinguish them. Additional scoring algorithms can remedy
this by providing confidence metrics in addition to these search
results, reducing ambiguity. Here we describe challenges to interpreting
phosphoproteomics data and review several different approaches to
determine sites of phosphorylation for both data-dependent and
data-independent acquisition-based workflows. Finally, we discuss open
questions regarding neutral losses, gas-phase rearrangement, and false
localization rate estimation experienced by both types of acquisition
workflows and best practices for managing ambiguity in phosphosite
determination.