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There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N + = 70 amu), fragments of the sulfurproducing cystathionine-containing enzymes, and the Ag ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N + amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfurproducing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the labelfree identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory. N insights into the function and activity of biological systems, from the toxicity of nanomaterials to the role of misfolded proteins in disease, require not only the identification of the proteins and nanomaterials but also knowledge of their localization and chemistry within complex biological assemblies. This characterization must relate the spatial distribution of the proteins or particles of interest to their cellular surroundings. Traditionally, analyte labeling (e.g., with fluorescent dyes or radioisotopes) is used to image the distribution of protein or enzymes; however, a limitation of fluorescence microscopy is that it relies on a prior knowledge of which proteins are present in a specific tissue; in addition, the spatial resolution of this technique is diffraction-limited. Super resolution light microscopy surpasses the diffraction limit and is a very powerful tool for imaging proteins inside cells; nevertheless, advanced knowledge of which proteins are present is still required. An alternative method for spatially resolved, label-free surface analysis of enzymes and proteins within cells is timeof-flight secondary ion mass spectrometry (TOF-SIMS). In TOF-SIMS, the sample surface is bombarded by an energetic focused primary ion beam leading to the ejection of secondary ions. The energy of the primary ion beam will vary depending on the instrumentation used and may be as low as sub-keV or as high as MeV. These ions (positive or negative) are accelerated by a potential of 2000 V into the flight tube. The Received: April 8, 2019 Accepted: July 16, 2019 Published: July 16, 2019 Article pubs.acs.org/ac Cite This: Anal. Chem. 2019, 91, 11098−11107 © 2019 American Chemical Society 11098 DOI: 10.1021/acs.analchem.9b01704 Anal. Chem. 2019, 91, 11098−11107 D ow nl oa de d vi a C O L U M B IA U N IV o n O ct ob er 2 2, 2 01 9 at 0 9: 45 :2 7 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s.

Label-Free Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Sulfur-Producing Enzymes inside Microglia Cells following Exposure to Silver Nanowires