Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix-assisted laser desorption/ ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed.However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on-tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD−MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.

The tumor suppressor p53 is the most mutated protein in human cancers. It is implicated in lung (70 %), colon (60 %), and stomach (45 %) cancers, respectively. [1] The latest release (R 16) of the International Agency for Research on Cancer (IARC) TP53 mutation database contains 29575 somatic mutations (November 2012; http://www-p53.iarc.fr/). A distinctive feature of the p53 mutational map is the rate of occurrence of missense mutations. Indeed, these single-point amino acid substitutions in p53 lead to abrogation of protein function, rather than deletions or nonsense mutations, as it is the case with most tumour suppressor proteins. A technique that is able to rapidly distinguish p53 mutants at low concentrations could have marked benefits for cancer screening assays and also for drug discovery. In this study, we used ion-mobility mass spectrometry (IM-MS) for this task.The p53 protein contains 393 amino acids and is divided into several structural and functional domains (Figure 1 a): a transactivation domain (TAD, residues 1-61, a proline-rich fragment (PR, residues 62-94) with multiple copies of the PXXP sequence, a DNA-binding domain (DBD, residues 94-292), a tetramerization domain (TET, residues 325-355), and a strongly basic C-terminal regulatory domain (CT, residues 363-393). [2] Very few mutations have been reported in the Nor C-terminal domains. [3] The central core of the protein, consisting of the DNA-binding domain, is the most highly conserved domain and is required for sequence-specific DNA binding. The majority of tumour-derived mutations (over 95 %) are mapped to the DBD, where the cluster of six socalled "hot spots" is located. [4] The structure of the p53 DBD was first solved by Cho et al. in 1994. [5] Based on the structure, the "hot spots" were classified as "structural" or "contact" mutations. These residues can affect either the thermodynamic stability and hence the structural integrity of the p53 DBD, or the conformation of the protein required for protein-DNA or protein-protein interactions. [2,[6][7][8][9][10][11] Herein, we report IM-MS studies on the conformational diversity of wild-type p53 and common cancer-associated p53 mutants. We define "conformational phenotypes" and monitor the variation in these as exhibited by four single-point mutations: R249A, R273H, K292I, and A276Y. Locations of mutated residues used in the studies are depicted schematically on the 3D structure of p53 (Figure 1 b). Specifically, we test whether the second-site suppressor mutant from loop L1, H115N, could trigger conformational changes in p53 cancerassociated mutations. In addition we use mass spectrometry as a tool to test the DNA-binding properties of the wild-type (WT) p53 and H115N mutant proteins.IM-MS can provide detailed insights into the structures of macromolecular systems. [12][13][14][15][16] Measured drift times are recorded as arrival-time distributions (ATDs), which can then be converted into collision cross sections (CCSs). [17,18] In this study, a Synapt HDMS [19] (Waters Corporation, Man-...

A series of rare earth complexes of the form Ln(LR)3 supported by bidentate ortho-aryloxide–NHC ligands are reported (LR = O(o-C6H2–tBu2-2,6-CN(C2H2)NR); R = iPr, tBu, Mes; Ln = Ce, Sm, Eu).

Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In LC–MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD–MALDI–MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD–MSI by adapting existing CD methods.