<div> Advancements in cancer treatments have increased rapidly in recent years, but cures remain elusive. Surgical tumor resection is a central treatment for many solid malignancies. Residual tumor at surgical margins leads to tumor recurrence. Novel tools for assessing residual tumor at surgical margins could improve surgical outcomes by helping to maximize the extent of resection. Ambient ionization-mass spectrometry (MS) methods generate and analyze ions from minimally prepared samples in near-real-time (e.g. seconds to minutes). These methods leverage the high sensitivity and specificity of mass spectrometry for analyzing gas phase ions and generating those ions quickly and with minimal sample preparation. Recent work has shown that differential profiles of ions, corresponding to phospholipids and small metabolites, are detected from cancerous and their respective normal tissue with ambient ionization-MS methods. When properly implemented, ambient ionization-MS could be used to assess for tumor at surgical margins and provide a molecular diagnosis during surgery. </div><div><br></div><div>The research herein reports efforts in developing rapid intraoperative ambient ionization-MS methods for the molecular assessment of cancerous tissues. Touch spray (TS) ionization and desorption electrospray ionization (DESI) were utilized to analyze kidney cancer and brain cancer.</div><div><br></div><div> As a demonstration of the applicability of TS-MS to provide diagnostic information from fresh surgical tissues, TS-MS was used to rapidly analyze renal cell carcinoma and healthy renal tissue biopsies obtained from human subjects undergoing nephrectomy surgery. Differential phospholipid profiles were identified using principal component analysis (PCA), and the significant ions were characterized using multiple stages of mass spectrometry and high resolution/exact mass MS. The same TS-MS analyzed renal tissues were subsequently analyzed with DESI-MS imaging to corroborate the TS-MS results, and the significant DESI-MS ions were also characterized with MS.</div><div><br></div><div>Significant efforts were made in developing and evaluating a standalone intraoperative DESI-MS system for analyzing brain tissue biopsies during brain tumor surgery. The intraoperative DESI-MS system consists of a linear trap quadrupole mass spectrometer placed on a custom-machined cart that contains all hardware for operating the mass spectrometer. This instrument was operated in the neurosurgical suites at Indiana University School of Medicine to rapidly analyze brain tissue biopsies obtained from glioma resection surgeries. A DESI-MS library of normal brain tissue and glioma was used to statistically classify the brain tissue biopsies collected in the operating room. Multivariate statistical methodologies were employed to predict the disease state and tumor cell percentage of the samples. A DESI-MS assay for detecting 2-hydroxyglutarate (2HG), the oncometabolic product of the isocitrate dehydrogenase (IDH) mutation (a key glioma prognostic marker), was developed and applied to determine the IDH mutation status during the surgical resection. The strengths, weaknesses, and areas of future work in this field are discussed. </div><div><br></div>