Enhanced Formation of Multiply Charged Protein and Peptide Ions in SIMS Using Large Water Clusters

Protein detection in secondary ion mass spectrometry (SIMS) has historically been limited by extensive fragmentation and low ionization yields. The introduction of large water gas cluster ion beams (GCIB) has significantly mitigated molecular fragmentation and enhanced ionization efficiency due to the large size and proton-rich nature of the primary ions, respectively. In this study, we demonstrate the successful application of water clusters for analyzing molecular ions of ubiquitin and angiotensin II. Compared to (Ar/CO₂)_n clusters, water clusters produced substantially higher ion yields for these molecules. However, when a matrix was used, the ion yields for water clusters decreased, while an increase was observed for (Ar/CO₂)_n clusters. Additionally, increasing the cluster energy led to corresponding increases in ion yield at equivalent energy per nucleon (E/m) values. Notably, water clusters facilitated the generation of multiply charged species of both ubiquitin and angiotensin II, including the detection of 3+ and 4+ charge states for ubiquitin. This ability to form and detect multiply charged protein species represents a major advancement for SIMS analysis, potentially expanding its applicable mass range. Furthermore, these findings have promising implications for high-resolution, multimodal mass spectrometry imaging that incorporates protein detection and analysis with no need for sample preprocessing or matrix addition.