DDA‐imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer

Matrix‐assisted laser desorption/ionization‐mass spectrometry imaging (MALDI‐MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA‐imaging technology where high‐resolution MSI is combined with parallel acquisition of lipid tandem MS(2) spectra on a hybrid ion trap‐Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA‐imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA‐imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA‐imaging on the Orbitrap Elite. Altogether, our work shows that DDA‐imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.