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Close deposition of the sample and external standard was used in axial matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200 μm wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5 mm × 57.0 mm by varying the gap between the traces from 100 μm to 4 mm. During acquisition, two adjacent traces were alternately irradiated with a 200‐Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide …