The emergence of imaging mass spectrometry in drug discovery and development: Making a difference by driving decision making.

The pharmaceutical industry is a dynamic, science-driven business constantly under pressure to innovate and morph into a higher performing organization. Innovations can include the implementation of new technologies, adopting new scientific methods, changing the decision-making process, compressing timelines, or making changes to the organizational structure. The drivers for the constant focus on performance improvement are the high cost of R&D as well as the lengthy timelines required to deliver new medicines for unmet needs. Successful innovations are measured against both the quality and quantity of potential new medicines in the pipeline and the delivery to patients. In this special feature article, we share our collective experience implementing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technology as an innovative approach to better understand the tissue biodistribution of drugs in the early phases of drug discovery to establish pharmacokinetic-pharmacodynamic (PK-PD) relationships, as well as in the development phase to understand pharmacology, toxicology, and disease pathogenesis. In our experience, successful implementation of MALDI IMS in support of therapeutic programs can be measured by the impact IMS studies have on driving decision making in pipeline progression. This provides a direct quantifiable measurement of the return to the organization for the investment in IMS. We have included discussion not only on the technical merits of IMS study conduct but also the key elements of setting study objectives, building collaborations, data integration into the medicine progression milestones, and potential pitfalls when trying to establish IMS in the pharmaceutical arena. We categorized IMS study types into five groups that parallel pipeline progression from the earliest phases of discovery to late stages of preclinical development. We conclude the article with some perspectives on how we see MALDI IMS maintaining relevance and becoming further embedded as an essential tool in the constantly changing environment of the pharmaceutical industry.

Imaging mass spectrometry reveals direct albumin fragmentation within the diabetic kidney.

Albumin degradation in the renal tubules is impaired in diabetic nephropathy such that levels of the resulting albumin fragments increase with the degree of renal injury. However, the mechanism of albumin degradation is unknown. In particular, fragmentation of the endogenous native albumin has not been demonstrated in the kidney and the enzymes that may contribute to fragmentation have not been identified. To explore this we utilized matrix-assisted laser desorption/ionization imaging mass spectrometry for molecular profiling of specific renal regions without disturbing distinct tissue morphology. Changes in protein expression were measured in kidney sections of eNOS-/-db/db mice, a model of diabetic nephropathy, by high spatial resolution imaging allowing molecular localizations at the level of single glomeruli and tubules. Significant increases were found in the relative abundances of several albumin fragments in the kidney of the mice with diabetic nephropathy compared with control nondiabetic mice. The relative abundance of fragments detected correlated positively with the degree of nephropathy. Furthermore, specific albumin fragments accumulating in the lumen of diabetic renal tubules were identified and predicted the enzymatic action of cathepsin D based on cleavage specificity and in vitro digestions. Importantly, this was demonstrated directly in the renal tissue with the endogenous nonlabeled murine albumin. Thus, our results provide molecular insights into the mechanism of albumin degradation in diabetic nephropathy.

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis.

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection.

Non-derivatized glycan analysis by reverse phase liquid chromatography and ion mobility-mass spectrometry.

A simple method for the analysis of non-derivatized glycans using a reverse phase column on a liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) instrument. The methodology supports both glycomic and proteomic work flows without the necessity of switching columns.

Bond-specific dissociation following excitation energy transfer for distance constraint determination in the gas phase.

Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase.

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer.

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohydrates), which enables structural comparisons to be made between molecules of different chemical compositions for the rapid "omni-omic" characterization of complex biological samples. Comparisons made between helium and nitrogen-derived CCS measurements demonstrate that nitrogen CCS values are systematically larger than helium values; however, general separation trends between chemical classes are retained regardless of the drift gas. These results underscore that, for the highest CCS accuracy, care must be exercised when utilizing helium-derived CCS values to calibrate measurements obtained in nitrogen, as is the common practice in the field.