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Rationale: As a powerful ambient ion source, atmospheric pressure (AP) matrix‐assisted laser desorption/ionization (MALDI) enables direct analysis at atmospheric pressure/temperature and minimal sample preparation. With the increasing usage of AP‐MALDI sources with Orbitrap instruments, systematic characterization of the extent of ion suppression effect (ISE) in AP‐MALDI‐Orbitrap mass spectrometry imaging (MSI) is desirable. Recently, a new low‐pressure MALDI platform has been introduced that reportedly provided better sensitivity. While extensive research efforts have been devoted to improving spatial resolution, fewer studies focused on the characterization and sensitivity improvement of these MALDI platforms that, coupled with high‐resolution Orbitraps, provide powerful strategy for MSI. 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. [ABSTRACT FROM AUTHOR]

Comprehensive protein identification and concomitant structural probing of proteins are of great biological significance. However, this is challenging to accomplish simultaneously in one confined space. Here, we develop a nanosecond photochemical reaction (nsPCR)-based click chemistry, capable of structural probing of proteins and enhancing their identifications through on-demand removal of surrounding matrices within nanoseconds. The nsPCR is initiated using a photoactive compound, 2-nitrobenzaldehyde (NBA), and is examined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Benefiting from the on-demand matrix-removal effect, this nsPCR strategy enables enhanced neuropeptide identification and visualization from complex tissue samples such as mouse brain tissue. The design shows great promise for structural probing of proteins up to 155 kDa due to the exclusive accessibility of nsPCR to primary amine groups, as demonstrated by its general applicability using a series of proteins with various lysine residues from multiple sample sources, with accumulated labeling efficiencies greater than 90%. Mass spectrometry-based quantitative proteomics aim to identify and quantify proteins from complex biological samples. Here, the authors developed a method for simultaneous high-throughput protein labelling and on-demand matrix removal within nanoseconds. [ABSTRACT FROM AUTHOR]