In the present work we describe the principles of operation, versatility and applicability of a trapped ion mobility spectrometer (TIMS) analyzer for fast, gas-phase separation of molecular ions based on their size-to-charge ratio. Mobility-based separation using a TIMS device is shown for a series for isobar pairs. In a TIMS device, mobility resolution depends on the bath gas velocity and analysis scan speed, with the particularity that the mobility separation can be easily tuned from low to high resolution (R>50) in accordance with the analytical challenge. In contrast to traditional drift tube IMS analyzer, a TIMS device can be easily integrated in a mass spectrometer without a noticeable loss in ion transmission or sensitivity, thus providing a powerful separation platform prior to mass analysis.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolutions and 20 pixels/s throughout molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 ×10 μm at the sample surface. General image performance was assessed using murine kidney and brain *