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RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial to understand the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution Ion Mobility Spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry (MS).METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques including linear cyclic IMS (cIMS), linear Trapped IMS (TIMS), and nonlinear high-Field Asymmetric Waveform IMS (FAIMS) were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue.RESULTS: Direct infusion of GT derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS were discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research.CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

Mass spectrometry imaging allows for the presence and abundance of a wide range of bio-molecules to be measured directly from tissue sections without the use of labels or tags. This results in the ability to compare the spatial distribution of hundreds of metabolites, lipids, peptides, and proteins within the same section in a single experiment. Desorption electrospray ionization (DESI) mass spectrometry imaging can differentiate and classify tissue types, and associated disease states, by measuring the unique characteristic molecular fingerprints of the tissues1,2. Furthermore, using histologically annotated databases of molecular profiles, unknown tissues can be classified using the mass spectrometry output coupled to machine learning approaches. A major benefit of DESI is that the analyzed sections are left largely unchanged by the process; the sections can subsequently be stained and the results validated by immunohistochemistry or conventional H&E approaches. During DESI fingerprinting analyses, a full mass spectrum on a cell by cell resolution of 20-50µm pixel size is collected. Chemical changes on very small scales can be distinguished and explored, allowing the chemistry of tumor margins and interface zones to be understood through mapping results onto metabolic pathways. As the data in a DESI experiment is collected and stored, multiple interrogations of the data are possible; as new discoveries are made, previously collected data can be retrospectively mined for additional insights. In this study, the three-dimensional chemical heterogeneity of drug dosed spheroids and tumor xenografts was measured with DESI and compared with MRI and PET imaging modalities and with FT-IR spectroscopic techniques. We demonstrate the power of this multi-analytical approach in glioma models and in colorectal and prostrate tumor samples to uncover metabolic and lipidomic changes during disease progression and to reveal the inherent heterogeneity within a tumor. 1. 1. Calligaris, D.Caragacianu, D.Liu, et al; PNAS, 2014, 111, 15185-151892. Guenther, S., Muirhead, L.J., et al, Cancer Res, 2015, 75, 1828-1837 Citation Format: Emrys A. Jones, Dipa Gurung, Fiona Henderson, Matthew Gentry, Danielle McDougall, Yasmin Shanneik, James Langridge, Adam McMahon, Zoltan Takats. Label free molecular imaging of tumor sections for two and three dimensional tissue classification and pathway mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4534.

Gas-phase protein separation by ion mobility: With its ability to separate the Parkinson's disease protein Alpha-synuclein and its autoproteolytic products—despite the small concentrations of the latter—ion-mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization-aggregation. In particular, a possible key fragment, the highly aggregating C-terminal fragment, AlphaSyn(72–140), has been revealed.

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250−300 proteins per 500 ng of tissue with 1D LC−MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10−15). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors., We report a cost-effective, single tube extraction methodology permitting the quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction from FFPE tissue blocks. In combination with a liquid chromatography−mass spectrometry-based label-free quantitation, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation of the methodology in renal cancer tissues is presented.

Mitochondria play a crucial role in cellular homeostasis, which justifies the increasing interest in mapping the different components of these organelles. Here we have focused our study on the identification of proteins of the mitochondrial inner membrane (MIM). This membrane is of particular interest because, besides the well known components of the respiratory chain complexes, it contains several ion channels and many carrier proteins that certainly play a key role in mitochondrial function and, therefore, deserve to be identified at the molecular level. To achieve this goal we have used a novel approach combining the use of highly purified mouse liver mitochondrial inner membranes, extraction of membrane proteins with organic acid, and two-dimensional liquid chromatography coupled to tandem mass spectrometry. This procedure allowed us to identify 182 proteins that are involved in several biochemical processes, such as the electron transport machinery, the protein import machinery, protein synthesis, lipid metabolism, and ion or substrate transport. The full range of isoelectric point (3.9-12.5), molecular mass (6-527 kDa), and hydrophobicity values (up to 16 transmembrane predicted domains) were represented. In addition, of the 182 proteins found, 20 were unknown or had never previously been associated with the MIM. Overexpression of some of these proteins in mammalian cells confirmed their mitochondrial localization and resulted in severe remodeling of the mitochondrial network. This study provides the first proteome of the MIM and provides a basis for a more detailed study of the newly characterized proteins of this membrane.

As part of our studies on the structure of yeast tRNAfMet, we investigated the incorporation of N-{[9-(β-D-ribofuranosyl)-9H-purin-6-yl]carbamoyl}-L-threonine (t6A) in the loop of a RNA 17-mer hairpin. The carboxylic function of the L-threonine moiety of t6A was protected with a 2-(4-nitrophenyl)ethyl group, and a (tert-butyl)dimethylsilyl group was used for the protection of its secondary OH group. The 2′-OH function of the standard ribonucleotide building blocks was protected with a [(triisopropylsilyl)oxy]methyl group. Removal of the base-labile protecting groups of the final RNA with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then with MeNH2 was done under carefully controlled conditions to prevent hydrolysis of the carbamate function, leading to loss of the L-threonine moiety.

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of α,β-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3′- and 5′-monophosphate; 3′,5′-bis-phosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans. © 1998 John Wiley & Sons, Ltd.

This paper shows the use of a quadrupole time-of-flight mass spectrometer combined with a liquid chromatograph for the identification of trace impurities in a drug substance. LC/MS/MS data obtained on trace impurities using this instrument compare favourably with those previously obtained on triple quadrupole mass spectrometers and in addition the high resoltuion capabilities of the ToF analyser allow accurate mass measurement of the fragment ions to better than 5 ppm in most cases. © 1997 John Wiley & Sons, Ltd.

The application of fast atom bombardment mass spectrometry (MS) and collisionally induced dissociation with mass-analysed kinetic energy spectrum scanning to biochemical studies of cyclic nucleotides is reviewed. MS analysis of partially purified tissue extracts has enabled the demonstration of the natural occurrence in mammals and higher plants of five cyclic nucleotides in addition to the previously established adenosine 3′,5′-cyclic monophosphate; analysis of the products of a putative cytidylate cyclase enzyme-catalyzed reaction has demonstrated the biosynthesis of cytidine 3′,5′-cyclic monophosphate and four novel analogues as side products, thereby enabling the development of specific assays. Quantitative applications have included kinetic analysis of phosphodiesterase and cyclic nucleotide-responsive protein kinase activity, while further quantitative applications have included the structural elucidation of synthetic cyclic nucleotide derivatives used in pharmacological studies. The impact of MS upon cyclic nucleotide research and its future potential are discussed.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.

The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.