High-Throughput Deconvolution of Native Protein Mass Spectrometry Imaging Data Sets for Mass Domain Analysis

[Image: see text] Protein mass spectrometry imaging (MSI) with electrospray-based ambient ionization techniques, such as nanospray desorption electrospray ionization (nano-DESI), generates data sets in which each pixel corresponds to a mass spectrum populated by peaks corresponding to multiply charged protein ions. Importantly, the signal associated with each protein is split among multiple charge states. These peaks can be transformed into the mass domain by spectral deconvolution. When proteins are imaged under native/non-denaturing conditions to retain non-covalent interactions, deconvolution is particularly valuable in helping interpret the data. To improve the acquisition speed, signal-to-noise ratio, and sensitivity, native MSI is usually performed using mass resolving powers that do not provide isotopic resolution, and conventional algorithms for deconvolution of lower-resolution data are not suitable for these large data sets. UniDec was originally developed to enable rapid deconvolution of complex protein mass spectra. Here, we developed an updated feature set harnessing the high-throughput module, MetaUniDec, to deconvolve each pixel of native MSI data sets and transform m/z-domain image files to the mass domain. New tools enable the reading, processing, and output of open format .imzML files for downstream analysis. Transformation of data into the mass domain also provides greater accessibility, with mass information readily interpretable by users of established protein biology tools such as sodium dodecyl sulfate polyacrylamide gel electrophoresis.