Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complimentary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum.

RNA hybridization‐based spatial transcriptomics provides unparalleled detection sensitivity. However, inaccuracies in segmentation of image volumes into cells cause misassignment of mRNAs which is a major source of errors. Here, we develop JSTA, a computational framework for joint cell segmentation and cell type annotation that utilizes prior knowledge of cell type‐specific gene expression. Simulation results show that leveraging existing cell type taxonomy increases RNA assignment accuracy by more than 45%. Using JSTA, we were able to classify cells in the mouse hippocampus into 133 (sub)types revealing the spatial organization of CA1, CA3, and Sst neuron subtypes. Analysis of within cell subtype spatial differential gene expression of 80 candidate genes identified 63 with statistically significant spatial differential gene expression across 61 (sub)types. Overall, our work demonstrates that known cell type expression patterns can be leveraged to improve the accuracy of RNA hybridization‐based spatial transcriptomics while providing highly granular cell (sub)type information. The large number of newly discovered spatial gene expression patterns substantiates the need for accurate spatial transcriptomic measurements that can provide information beyond cell (sub)type labels.

<p>Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complimentary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum.</p>

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