Conclusions
The new SubAP-MALDI source coupled with an Orbitrap mass spectrometer was established as a viable platform for in situ endogenous biomolecular analysis with increased sensitivity compared with conventional AP-MALDI sources as evidenced by the confident identification of neuropeptides from mouse brain imaging analyses. The alleviated ISE was key to substantial performance improvement due to optimized intermediate pressure conditions and better ion collection by the ion funnel.
Methods
We compared the analytical performance of AP and low-pressure (subatmospheric) MALDI sources to study the effect of pressure control in the ion source. Using a model peptide/protein mixture, we systematically evaluated the factors influencing ISE. Furthermore, the effect of laser spot size was evaluated through tissue imaging analysis of lipids and neuropeptides. The effects of ion suppression and laser spot size have also been examined by comparing the number of identified molecular species during MSI analysis.
Results
Several key operating parameters including source pressure, laser energy, laser repetition rate, and microscopic slide coating materials were optimized to minimize the ISE. Under the optimal conditions, the subatmospheric AP-MALDI-Orbitrap platform with high spatial and mass spectral resolution enabled significantly improved coverage of several lipid and neuropeptide families in the MS analysis of mouse brain tissue sections. Conclusions: The new SubAP-MALDI source coupled with an Orbitrap mass spectrometer was established as a viable platform for in situ endogenous biomolecular analysis with increased sensitivity compared with conventional AP-MALDI sources as evidenced by the confident identification of neuropeptides from mouse brain imaging analyses. The alleviated ISE was key to substantial performance improvement due to optimized intermediate pressure conditions and better ion collection by the ion funnel.