Multicenter Validation Study of Quantitative Imaging Mass Spectrometry.

The aim of this study was to assess potential sources of variability in quantitative imaging mass spectrometry (IMS) across multiple sites, analysts, and instruments. A sample from rat liver perfused with clozapine was distributed to three sites for analysis by three analysts using a predefined protocol to standardize the sample preparation, acquisition, and data analysis parameters. In addition, two commonly used approaches to IMS quantification, the mimetic tissue model and dilution series, were used to quantify clozapine and its major metabolite norclozapine in isolated perfused rat liver. The quantification was evaluated in terms of precision and accuracy with comparison to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The results of this study showed that, across three analysts with six replicates each, both quantitative IMS methods achieved relative standard deviations in the low teens and accuracies of around 80% compared to LC-MS/MS quantification of adjacent tissue sections. The utility of a homogeneously coated stable-isotopically labeled standard (SIL) for normalization was appraised in terms of its potential to improve precision and accuracy of quantification as well as qualitatively reduce variability in the sample tissue images. SIL normalization had a larger influence on the dilution series, where the use of the internal standard was necessary to achieve accuracy and precision comparable to the non-normalized mimetic tissue model data. Normalization to the internal standard appeared most effective when the intensity ratio of the analyte to internal standard was approximately one, and thus precludes this method as a universal normalization approach for all ions in the acquisition.

Metabolism of [(14)C]GSK977779 in rats and its implication with the observed covalent binding.

GSK977779 is a potent HM74a agonist evaluated for the treatment of dyslipidemia. The disposition and metabolism of [(14)C]GSK977779 (67.6 µmol/kg p.o.) was studied in male and female rats. The compound was well absorbed and its primary route of elimination was in the feces. Based on metabolite profiling of plasma extracts and urine and bile samples, it was demonstrated that GSK977779 was extensively metabolized in the rat by N-dealkylation, mono- and dioxygenation, reductive and oxidative cleavage of the 1,2,4-oxadiazole ring, and conjugative pathways. After plasma extraction high amounts of nonextractable radioactivity were observed, which were more pronounced in female rats. Size-exclusion chromatography and SDS gel electrophoresis indicated that the majority of the nonextractable radioactivity was covalently bound to plasma proteins. Solubilization of the plasma protein pellet followed by high-performance liquid chromatography and mass spectrometry suggested that a carboxylic acid metabolite derived from oxadiazole ring cleavage may be responsible for the observed covalent binding of the radioactivity to rat plasma proteins.

Protein signatures for survival and recurrence in metastatic melanoma.

Patients with melanoma metastatic to regional lymph nodes exhibit a range in tumor progression, survival, and treatment. Current approaches to stratify patients with this stage of disease predominantly involve clinical and histological methods. Molecular classification thus far has focused almost exclusively on genetic mutations. In this study, proteomic data from 69 melanoma lymph node metastases and 17 disease free lymph nodes acquired by histology-directed MALDI imaging mass spectrometry were used to classify tumor from control lymph node and to molecularly sub-classify patients with stage III disease. From these data, 12 survival associated protein signals and 3 recurrence associated signals in the acquired mass spectra were combined to generate a multiplex molecular signature to group patients into either poor or favorable groups for recurrence and survival. Proteins represented in the signature include cytochrome c, s100 A6, histone H4, and cleaved forms of thymosin ß-4, thymosin ß-10, and ubiquitin. In total over 40 protein signals from the tissue were identified.

Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry.

A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain.