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Cholesterol serves as a biomarker in clinical- and life-sciences. The determination of abnormal levels can indicate several types of human diseases. However, the low polarity of free cholesterol makes it hardly accessible by (nano) electrospray ionization mass spectrometry (nESI-MS). As novel approach, the flexible microtube plasma (FμTP) for post-ionization allows the determination of low-polar compounds like cholesterol in combination with nESI-MS. Focusing on the analytical performance, the activated post-ionization leads to an increased cholesterol signal by a factor of 22. The repeatability and long-term stability could be successful evaluated by using a complex liver extract. Via the method of standard addition, a linear dynamic range of 1.7 orders of magnitude, a minimum detectability of 3.71 mg/L and a high accuracy (deviation: − 8.11 %) is demonstrated proofing the FμTP-nESI-MS as an excellent approach for a derivatization-free determination of cholesterol without the necessity of high-resolution Orbitrap devices or enhanced MS acquisition-methods.

Previously published data have shown an allele-specific variation in the in vitro binding of apolipoprotein E (apoE) to tau, which prompted the hypothesis that apoE binding may protect tau from phosphorylation, apoE3 being more efficient than apoE4. We have, therefore, investigated the effects of apoE on tau phosphorylation in vitro by the proline-directed kinase, glycogen synthase kinase (GSK)-3β. The phosphopeptide maps of tau alone, of tau with apoE3 and of tau with apoE4 were very similar. When apoE2 was present a further four spots were evident. Additionally, of the 15 peptides phosphorylated in the presence or absence of apoE, subtle differences, some isoform-specific, in the relative amounts of phosphorylation were observed.

Binding of three ruthenium(ii) compounds of general formula mer-[Ru(L3)(N-N)X][Y] (where L3 = 4-chloro-2,2:6,2-terpyridine (Cl-tpy); N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach) or 2,2-bipyridine (bipy); X = Cl; Y = Cl) to human serum albumin (HSA) has been investigated by nano-LC/nano-ESI MS and docking studies. A bottom-up proteomics approach has been applied for the structural characterization of metallated proteins and the data were analyzed in both the positive and negative ion mode. The negative ion mode was achieved after the post-column addition of an isopropanol solution of formaldehyde that enabled sample ionization at micro-flow rates. The negative ion mode MS has been proved to be beneficial for the analysis of binding sites on ruthenated protein in terms of ion charge reduction and consequent simplification of target sequence identification based on isotopic differences between ruthenated and non-ruthenated peptides. Moreover, the negative ion mode ESI MS shows the advantage of singly charged ion formation and, unlike MALDI MS, it does not cause complete ligand fragmentation, merging the benefits of each method into a single experiment. Six target sequences were identified for the binding of en and dach compounds, and four sequences for the binding of bipy. All compounds have been found to bind histidine and one aspartate residue. Docking studies showed that the identified sequences are the constituents of five distinct binding sites for en and dach, or two sites for the bipy complex. The selection of binding sites seems to be dependent on the chelate ligand and the form of the complex prior or after hydrolysis of the leaving chloride ligand. This is a pre-copyedited, author-produced version of an article accepted for publication in Metallomics following peer review. The version of record Nišavić, M.; Janjić, G. V.; Hozić, A.; Petković, M.; Milčić, M. K.; Vujčić, Z.; Cindrić, M. Positive and Negative Nano-Electrospray Mass Spectrometry of Ruthenated Serum Albumin Supported by Docking Studies: An Integrated Approach towards Defining Metallodrug Binding Sites on Proteins. Metallomics 2018, 10 (4), 587–594 is available online at: [https://doi.org/10.1039/c7mt00330g]

Polynuclear hydroxide complexes play an important role for the hydrolysis of tetravalent thorium ions in aqueous solution, in particular for Th(IV) concentrations exceeding some [Th(IV)]=10−4 M. Consequently, these polymers must be considered when describing hydrolysis of Th(IV) or dissolution processes of Th(IV) solids. In the past, considerable efforts were made to obtain equilibrium formation constants of these polymers and different stoichiometries for dimers, tetramers and hexamers have been suggested. However, most information was obtained from indirect methods, in particular, from potentiometric titrations. In the present work, we present an approach of directly quantifying polymeric metal hydroxide complexes in solution. By nano-electrospray mass-spectrometry the degrees of polymerization, i.e. the numbers of Th4+ ions and the numbers of hydroxide ligands, and as a consequence, also the charges of the complexes are measured. All mono- and polynuclear species which are present in solution are quantified simultaneously down to species contributing less than 0.1% of the total [Th(IV)] concentration. Solutions of [Th(IV)]=6×10−6–10−1 M are investigated in HCl at [H+]=10−4–0.1 M. More than 30 different polymeric complexes are observed with the general trend of increasing number of hydroxide ligands with decreasing acidity. A surprising finding is the presence of the pentamer Th5(OH)y z +, which was not described in the literature before. With decreasing Th(IV) concentration the stability field of polymers narrows continuously until polymers can no longer be detected below [Th(IV)]=10−5 M.

An automated surface-sampling technique called liquid extraction surface analysis (LESA), coupled with infusion nano-electrospray high-resolution mass spectrometry and tandem mass spectrometry (MS/MS), is described and applied to the qualitative determination of surface chemical residues resulting from the artificial spraying of selected fresh fruits and vegetables with representative pesticides. Each of the targeted pesticides was readily detected with both high-resolution and full-scan collision-induced dissociation (CID) mass spectra. In the case of simazine and sevin, a mass resolution of 100 000 was insufficient to distinguish the isobaric protonated molecules for these compounds. When the surface of a spinach leaf was analyzed by LESA, trace levels of diazinon were readily detected on the spinach purchased directly from a supermarket before they were sprayed with the five-pesticide mixture. A 30 s rinse under hot running tap water appeared to quantitatively remove all remaining residues of this pesticide. Diazinon was readily detected by LESA analysis on the skin of the artificially sprayed spinach. Finally, incurred pyrimethanil at a level of 169 ppb in a batch slurry of homogenized apples was analyzed by LESA and this pesticide was readily detected by both high-resolution mass spectrometry and full-scan CID mass spectrometry, thus showing that pesticides may also be detected in whole fruit homogenized samples. This report shows that representative pesticides on fruit and vegetable surfaces present at levels 20-fold below generally allowed EPA tolerance levels are readily detected and confirmed by the title technologies making LESA-MS as interesting screening method for food safety purposes. Copyright © 2011 John Wiley & Sons, Ltd.

Glycans from six highly purified hFSH preparations were released by peptide-N-glycanase digestion and analyzed by negative mode nano-ESI mass spectrometry before and after neuraminidase digestion. Pituitary glycan structures were mainly high-mannose, di-, tri-, and tetra-antennary, and their abundance largely paralleled that reported by other investigators using different approaches. For most of the FSH preparations, the differences in glycosylation appeared to be restricted to relative abundances of the major glycan families, as defined by their neutral core oligosaccharide structures. Qualitative differences between glycan populations were largely relegated to those species that were lowest in abundance. Significant qualitative differences were noted in two cases. Recombinant GH3-hFSH triantennary glycans appeared to have the third antenna exclusively on the mannose6-branch, in contrast to all pituitary and urinary hFSH triantennary glycans, in which this antenna was exclusively attached to the mannose3-branch. The hypo-glycosylated hFSH preparation isolated from purified hLH was decorated with high mannose glycans that accounted for over 40% of the total in this population. As this preparation was found to be consistently 20-fold more active than hFSH24 in FSH receptor-binding assays, it appears that both macroheterogeneity and microheterogeneity in FSH preparations need to be taken into account.

Very few relevant methods have been described for the detection and quantitation of phenolic compounds in faecal matrix. Extraction with conventional organic solvents such as chloroform/methanol (2:1, Folch reagent), methanol and ethanol (72%) showed high extraction efficiency for lipids and also gave good recovery of the major phenolic compounds present in the matrix. However, in comparison with a newly developed phosphate buffer method, the yield of minor phenolics was negligible when detected by these conventional methods. Conventional methods also lead to contamination of the ion source of the mass spectrometer and rapid deterioration of column performance mostly due to the high concentration of lipids. However, if the faecal matrix is initially extracted with phosphate buffer, and the extract acidified and re-extracted with diethyl ether, the range and yield of phenolic compounds are enhanced and the problem of lipid contamination is substantially alleviated. Following pilot studies and optimisation of the procedure, individual phenolic compounds (n = 29) were identified by nano-electrospray ionisation mass spectrometry (nano-ESI-MS), nano-ESI-tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/EI-MS) and quantitated (n = 27) by GC/MS in subsets (n = 5) of faecal samples, collected during the European Agency for Cancer Prevention calcium/fibre intervention study from four European countries (Italy, Germany, Spain and Denmark). A range of phenolic compounds (mainly acids) was detected, dominated by phenylacetic, benzoic, phenylpropionic and m-hydroxyphenylpropionic acids, representing on average 9.91 (93%), 8.25 (92%), 9.45 (95%) and 11.05 (98%) mM in the Italian, German, Spanish and Danish samples, respectively. The new method should enable large epidemiologic, case-control and intervention studies on the relevance of phenolic antioxidants in the aetiology of colorectal cancer to be conducted in the future. Copyright © 2006 John Wiley & Sons, Ltd.

The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).

A new technique for accurate mass measurement utilizing multiple sprayer nano-electrospray ionization mass spectrometry (nano-ESI-MS) combined with nano-scale high-performance liquid chromatography (nano-HPLC) on a magnetic sector instrument is described. Both metal-coated glass capillaries and fused-silica capillaries were used as nano-ESI sprayers. A metal-coated glass capillary was used for the introduction of the Ref. compound solution, and a metal-coated fused-silica capillary was used for connection to the nano-HPLC column. By shifting each sprayer's position relative to the sampling orifice, spectra were obtained of both the sample components as eluted from the column and reference compounds. Several standard compounds were examined and satisfactory accurate masses were obtained. Problems arising from differences in ionization efficiency between the sample and reference compounds were not observed.

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.

A Fisons Quattro I electrospray ionization (ESI) source has been modified to produce stable electrospray ion currents at flow rates as low as 50 nL/min. The original counter electrode and skimmer cone lens of the Fisons ESI source have been replaced with a spherical cone lens. This improved source provides a greater range of x,y,z positioning of a stainless steel tip resulting in a stable ion signal for flow rates of 50 nL/min to 2 μL/min. A tapered stainless steel electrospray tip (50 μm i.d.) was evaluated for mass spectrometry using nano-liquid chromatography (50 μm i.d., flow rate = 120 nL/min) and sheathless capillary electrophoresis. The analysis of a nonionic surfactant, octylphenol ethoxylate, was accomplished with both nanoscale separation techniques. © 1998 John Wiley & Sons, Ltd.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

In earlier liquid chromatography/mass spectrometry (LC/MS) experiments using a commercially available nano-electrospray interface designed for the coupling of nano-LC (flow rate = 200 nL/min) to electrospray mass spectrometry, a sudden drop in the electrospray total ion current was observed at certain percentages of organic modifier in the mobile phase. Therefore the performance of this nano LC/MS system was evaluated for different mobile phase compositions. The uncoated, non-tapered fused silica tips (20 μm i.d.) which were delivered standard with the interface, and several other electrospray capillaries, were evaluated for different mobile phase compositions: uncoated, tapered (20→9 μm i.d.) fused silica tips; gold coated, tapered fused silica tips and stainless steel tips (70 μm i.d.). The use of tapered but uncoated fused silica tips did increase the performance of the nano-electrospray system. The best results were obtained with gold coated, tapered tips. Stainless steel tips with an i.d. of 70 μm gave no results at the applied flow rate of 200 nL/min. © 1998 John Wiley & Sons, Ltd.

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

The use of electrospray ionization mass spectrometry (ESI-MS) for studying non-covalent interactions between macromolecules and ligands is well established. ESI-MS can be a useful tool for the determination of dissociation constants between molecules in the gas phase. We validate this method by studying the binding of the catalytic domain of cellobiohydrolase I (CBH I) from Trichoderma reesei to the disaccharide inhibitor cellobiose. The method was further applied to study two newly synthesized cellobiose derivatives (m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside and p-benzyloxybenzyl beta-cellobioside). In a titration experiment, peak areas of different charge states of the free enzyme and the complex were summed in order to determine the dissociation constant. For cellobiose and m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside, the calculated values are in good agreement with those reported from either displacement titration or equilibrium binding experiments in solution. Due to non-specific binding, the dissociation constant of p-benzyloxybenzyl beta-cellobioside does not correspond with the solution-based value. Our results indicate the need for careful interpretation of data sets when using nanoESI to study non-covalent interactions.

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.

Neurosteroids are steroids synthesised in the central and peripheral nervous systems. Known neurosteroids include pregnenolone, dehydroepiandrosterone (DHEA), progesterone and its reduced metabolites. It has been demonstrated that neurosteroids modulate neurotransmission by binding to neurotransmitter receptors, and exert physiological functions that are clearly different from those of endocrine steroids. The effects of neurosteroids on improving the memory of cognitively impaired aged rats, on the inhibition of aggressiveness in castrated male mice, and trophic effects on neuronal regeneration and remyelination have been documented. The local synthesis, selective interaction with neurotransmitter receptors and behavioural effects of neurosteroids strongly suggests that they may have important physiological or pathophysiological roles. There is an increasing need to develop methods to analyse these hormones with high sensitivity and high specificity. In this thesis I focused on the development of methods combining nano-electrospray (ES) mass spectrometry with capillary column liquid chromatography (CLC) for the analysis of profiles of neurosteroids in rat brain. It was also an aim to make the methods applicable to a broad range of lipophilic biomolecules. Initially, synthetic steroid sulphates and unconjugated oxosteroids (ketosteroids) were studied by nano-ES and tandem mass spectrometry. Steroid sulphates could be detected as deprotonated molecules in full range scanned spectra at a level of 1 pg/µL. Information about steroid structure was obtained from collision-induced dissociation (CID) spectra of 1 ng of steroid sulphate, while characterisation of the sulphate ester group required only 3 pg of material. Unconjugated oxosteroids were converted into their oximes which were detected as protonated molecules with 20 times higher sensitivity than the underivatised steroids. The detection limits for the oximes of 3-oxo-delta4, 20-oxo and 17-oxo steroids were 2.5, 5, and 25 pg/µL, respectively in full range scans. CID spectra of the protonated oximes provided valuable information regarding the position of oxo and hydroxyl group(s). These studies established a basis for determination and structure characterisation of neurosteroids from brain samples. A procedure for CLC-ES mass spectrometry was then developed. A double splitter method was introduced which made it possible to use a pre-column for analyte focusing from large sample volumes. It also made it possible to operate the solvent pumps at flow rates compatible with gradient elution while the flow rates through the analytical column were compatible with micro-electrospray. The method was designed to be generally applicable to the analysis of biomolecules and its utilities were demonstrated by the analysis of steroid sulphates, in human plasma. In the course of these studies, certain CLC-ES conditions were found to cause on-column chemical transformations of 3beta- hydroxy-delta5 steroid sulphates. Radical species generated from electrolysis of water and methanol in the solvent are proposed to be responsible for the formation of oxidised and methoxylated products of these steroids. Other analytes with double bonds were also transformed under these conditions. Thus, on-column electrochemistry can be an important source of artefacts in analyses by CLC-ES mass spectrometry. The reactions could be prevented by appropriate grounding. The analysis of neurosteroids in rat brain required the development of an extraction, purification and subfractionation procedure. Brain steroids were extracted, and unconjugated neutral steroids and sulphated steroids were separated. The steroid sulphate fraction was then analysed by CLC-ES mass spectrometry. Endogenous sulphates of pregnenolone and DHEA were not detected at levels above the detection limit, 0.3 ng/g wet brain, while pregnenolone sulphate, added to brain extract at a level of 6.6 ng/g, was easily detected. The unconjugated oxosteroids were converted to their oximes, selectively isolated on a cation exchanger, and analysed by CLC-ES tandem mass spectrometry. The chromatograms showed the presence of progesterone, pregnenolone, pregnanolone isomers, DHEA and testosterone in rat brain. These steroids were characterised by tandem mass spectrometry, Based on the results of CLC-ES tandem mass spectrometry, the levels Of C21 and C19 steroids were estimated in the range of 0.04 - 20 ng/g wet brain. The levels of progesterone and testosterone showed a sex difference. During the development of the above analytical methods, nano-ES mass spectrometry was applied to the characterisation of a lipophilic modulatory factor isolated from mouse brain. The factor, which activated the retinoid X receptor (RXR), was extracted from mouse brain incubates, purified by HPLC and analysed by nano-ES and tandem mass spectrometry. Accurate mass measurement and CID spectra of the purified active compound revealed that it was cis-4,7,10,13,16,19-docosahexaenoic acid. In conclusion, the methods developed and described in this thesis are suitable for the analysis of sulphated steroids and oxosteroids, as well as other related compounds. With their high sensitivity the methods enable highly specific analysis of these important compounds from small amounts of sample.

Electrospray mass spectrometry is a standard tool for the investigation of biological samples. Due to the high flowrates of the standard sources, large sample amounts are required and it is almost impossible to spray physiological solutions due to their aqueous medium. The introduction of microelectrospray sources has made it possible to decrease the sample amounts needed and enabled the use of buffered solutions. In this work, a nanoES-like source based on a modification of an existing IonSpray source is introduced. In contrast to other nanoES sources available, the modification presented allows a fast change between the nanoES and the normal IonSpray modes. Copyright 2000 John WileySons, Ltd.

A new technique for accurate mass determination by using multiple sprayers nano-electrospray ionization mass spectrometry (nano-ESI-MS) on a magnetic sector instrument is described. Metal coated glass capillaries were used as nano-ESI sprayers. One of the sprayers was used for the reference compound solution, and others were used for the introduction of sample solutions. The spectra of the different compounds were obtained by shifting each sprayer's position relative to the sampling orifice. The accurate masses of several standard compounds were obtained with good accuracy, without problems arising from differences in ionization efficiency between the sample compounds and reference compound. Copyright © 2000 John Wiley & Sons, Ltd.

Publisher Summary Nano-electrospray (nES) is a new technique for characterizing biomolecules in small volumes (0.5–2 μl) at low picomole levels. In nES, signals from a single sample loading typically last more than 30 minutes, which permits optimization of instrument parameters and MS/MS sequencing with high sensitivity. Both nES/MS and nES/MS/MS data can be obtained from a single sample loading. These features make nES an attractive off-line technique for sequencing peptides collected from capillary electrophoresis (CE). In this chapter, an off-line approach that combines nES/MS analysis and Edman sequencing of peptide/protein fractions collected from CE is presented. Automatic peak collection is accomplished using a computer-controlled Beckman P/ACE 5000 instrument. An off-line approach that is simple and useful for peptide/protein sequencing using 5–10 picomoles of material has been demonstrated. Several different samples of material, including a peptide mixture (angiotensin-I, methionine enkephalin, and substance-P), a tryptic digest of cytochrome-C and proteins like myoglobin, insulin and lysozyme, are used to demonstrate this method. For this purpose, peptide and protein samples are first separated by capillary electrophoresis. Selected peaks are fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing.

Electrospray (ES) mass spectrometry has been used to analyse preparations of porcine pulmonary surfactant polypeptide-C (SP-C). A number of variant forms of the native 35-residue dipalmitoylated peptide were detected including (a) C-terminally methylated SP-C, (b) C-terminally methylated and methionine oxidized SP-C, (c) N-terminally truncated, C-terminally methylated and methionine oxidized SP-c, (d) C-terminally elongated, C-terminally methylated and methionine oxidized SP-C, and (e) tripalmitoylated, C-terminally methylated and methionine oxidized SP-C. C-terminal methylation and methionine oxidation are probably a consequence of the sample handling procedure. The occurrence of the C-terminally elongated form of SP-C has implications for the in vivo processing of proSP-C and the Tandem mass spectrometry (MS/MS) was used to confirm the amino acid sequence of SP-C and the presence of palmitoyl groups covalently linked to the peptide. Some of the structures of the variant forms of SP-C were determined by MS/MS.

Electrospray mass spectrometry is a standard tool for the investigation of biological samples. Due to the high flowrates of the standard sources, large sample amounts are required and it is almost impossible to spray physiological solutions due to their aqueous medium. The introduction of microelectrospray sources has made it possible to decrease the sample amounts needed and enabled the use of buffered solutions. In this work, a nanoES-like source based on a modification of an existing IonSpray source is introduced. In contrast to other nanoES sources available, the modification presented allows a fast change between the nanoES and the normal IonSpray modes. Copyright 2000 John Wiley & Sons, Ltd.

A novel multilayer modulator chip offering a robust miniaturized interface for multidimensional liquid chromatography has been developed. The thermoplastic microfluidic device comprises five tailor-made functional layers, and the chip is compatible with commercially available switching-valve technology. The modulator chip allows for robust ultrahigh-pressure operation up to 65 MPa. Peak-dispersion characteristics of system peaks were assessed directly at the valve outlet by monitoring fluorescein injection profiles with laser-induced fluorescence detection. Integration of a microporous monolithic mixing entity in the microchannels significantly narrows the resulting peak profile. Proof-of-concept of the applicability of the microfluidic modulator chip is demonstrated in a heart-cut multidimensional strong-cation-exchange-reversed-phase liquid chromatography proteomics analysis workflow coupled to nanoelectrospray mass spectrometry for the target analysis of Glu-1-Fibrinopeptide B spiked in a protein digest mixture of bovine serum albumin.

The mechanism of enhanced photocatalytic indole cation-radical cyclization in confined space is examined by coupling a nanopipette reactor with in situ nano-electrospray mass spectrometry (nanoESI-MS).

In this work, we report the coupling of solid-phase microextraction (SPME) enabled by surface-coated acupuncture needles with nano-electrospray mass spectrometry (nanoESI-MS) for the analysis of bioactive molecules in living plants. The needle tip was oxidized by a mixture of nitric acid and hydrogen peroxide solution and then subject to surface coating via carbonization of paraffin. A combination of oxidation and surface coating resulted in a thin coating of carbon film, whereby the significantly increased surface area promoted both analyte enrichment and ionization for MS analysis. The analytical performances were evaluated through the characterization of small molecules, peptides and proteins. Compared with conventional nanoESI, our new strategy of employing surface-coated needles had a high salt tolerance. The streamlined experimental workflow could be completed within one minute. The linear dynamic ranges for L-histidine and L-lysine, as two representatives, were over two orders of magnitude with a limit of detection (LOD) of 3.0~5.0 ng/mL. A mark is made on the needle at 2 mm from the tip, the needle is then kept in the sample for 30 s. In vivo sampling and identification of α-tomatine and organic acids from the stem of a living tomato plant were demonstrated as a practical application, while the physiological activities of the plant were not disrupted due to the minimally invasive sampling. We anticipate that the developed strategy may be of potential use for real-time clinical and other on-site analyses.

Experiments are described that illustrate solvent oxidation, emitter electrode corrosion, and analyte oxidation in positive ion mode nano-electrospray mass spectrometry using a wire-in-a-capillary, bulk-loaded nano-electrospray emitter geometry. Time-lapsed color photography of pH and metal specific indicator solutions within operating nano-electrospray emitters, as well as temporal changes in the ions observed in the nano-electrospray mass spectra, are used to probe these reactions, judge their magnitude, and study the time dependent changes in solution composition and gas-phase ion signal brought about as a result of these electrochemical reactions. The significance of these observations for analytical applications of nano-electrospray mass spectrometry are discussed. (J Am Soc Spectrom 2001, 853–862) Published by Elsevier Science Inc.

The self-assembly of intrinsically disordered protein tau into paired helical filament forms one of the hallmarks of Alzheimer's disease. However, the facets of innately disordered structure of tau and its conversion to a β-sheet-rich fibril during several tauopathies are poorly understood. Here, we provide a direct insight into the ensemble of highly heterogeneous conformational families of tau at physiological pH, by nano-electrospray mass spectrometry coupled with ion mobility. The average collision cross section of the most unfolded conformer was higher by >2 fold than that of the most folded one. Acidic pH largely induced unfolding in tau, obliterating the compact conformers completely. The highly unfolded conformers were the key species bestowing the unusual solubility to tau at low pH, with limited contribution from intramolecular long-range interfaces giving rise to ordered conformers. Contrarily, alkaline pH shifted tau towards folded conformations due to charge neutralization, keeping the overall random coil architecture intact. Intriguingly, the heparin-induced in vitro aggregation propensity of the protein attenuated at both acidic and alkaline pH, illustrating the significance of altered conformations in pathological functions of tau. Our observations at low pH indicate that a reorganization of the intricate network of momentary long-range contacts in tau might have implication in its aggregation pathology. Disease-modifying therapies for Alzheimer's disease targeting either to disrupt the essential fibril-forming interaction at third microtubule-binding repeat of tau or to perturb specific binding interaction of tau with endogenous polyanionic species might be of high benefit.

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that were able to activate p53 in ARN8 cells also largely inhibited DHODH. This yielded 12 other chemotypes capable of inhibiting DHODH. At this point we tested HZ00 analogues and identified a much more potent compound we named HZ05. HZ05 phenocopied HZ00 and demonstrated enantiomer-selective inhibition of DHODH with (R)- HZ05 inhibiting DHODH with an IC50 of 11 nM. We obtained a crystal structure of (R)- HZ05 in complex with DHODH and found that it occupied the same quinone tunnel as the known inhibitors brequinar and teriflunomide (A77 1726). HZ05 caused a number of tumour cells to accumulate in S-phase. We found that a slower cycling cell line, U2OS, required pretreatment with HZ05 to accumulate cells in S-phase prior to treatment with nutlin 3a to achieve tumour cell kill, as co-treatment resulted in G1 arrest. We therefore theorised that accumulating cells in S-phase with high levels of p53 predisposed them to cell kill upon application of a blocker of p53 degradation. The first sets of compounds found back in 2008 by the Laín laboratory were the tenovins. Tenovin 1 was the first compound identified from the screen, which used the T22 murine fibroblasts to establish its ability to activate p53 transcriptional activity in the reporter assay. Tenovin 1 was, however, not particularly soluble and therefore a more soluble analogue called tenovin 6 was synthesised. Tenovin 6 elicited many of the same cellular phenotypes as tenovin 1, and therefore target identification was conducted using tenovin 6. Tenovin 6 was subsequently identified as an inhibitor of SirT1 and SirT2 in a yeast genetic screen, biochemical assays and further target validation in mammalian cells. Tenovin 1 and 6 displayed a very similar profile – they both induced p53 transcriptional activity and both increased acetylation of both p53 and tubulin. This is where the similarity ends, however, as it was discovered, through extensive structure-activity relationship studies, that the targeting profiles of both molecules was markedly different. In Paper II we built upon previous studies that identified tenovin 6 as a compound capable of inhibiting autophagy. In this paper we conducted structure-activity relationships using tenovin analogues to understand the mechanism by which tenovins affect autophagy. We confirmed that tenovins capable of perturbing autophagy do so by inhibition of autophagic flux, in a similar manner to chloroquine, by raising the pH of lysosomes. We also isolated the portion of the molecule, a tertiary amine at the end of an aliphatic chain, as the reason for blockage of autophagic flux. Finally, we found that blockage of autophagic flux by tenovins is required to eliminate tumour cells in culture and that this blockage of autophagy is capable of killing mutant B-Raf tumour cells arrested in G1 by vemurafenib treatment. In Paper III we further explored the targeting profile of the tenovins and tested whether tenovins were capable of inhibiting DHODH. We found that tenovins 1 and 6 were capable of inhibiting DHODH at 113 nM and 500 nM respectively. We also conducted a thermal shift assay and identified tenovins 1, 6 and 39OH as being capable of interacting with DHODH in vitro. We then obtained a crystal structure of tenovin 6 occupying the same quinone tunnel as HZ05, brequinar and teriflunomide. Phenotypically, tenovin 1 and 33 had their ability to induce p53 transcriptional activity ablated upon addition of either uridine or orotate, but not dihydroorotate, whilst tenovin 6 had its ability to induce p53 transcriptional activity partially prevented by addition of uridine or orotate. Tenovin 39 and 39OH displayed no difference upon supplementation. Tenovin 1 and 33 also had their growth inhibitory effect markedly reduced upon orotate or uridine supplementation, but no other tenovin, including 6, showed any effect of supplementation. We also discovered another target of the tenovins – the ability to inhibit nucleoside uptake. We discovered that uridine uptake was blocked by tenovin 6, 33, 39, 39OH and 50. This paper, therefore, highlights the shifting targeting profile of the tenovins due to small molecular changes and that a phenotypic readout may remain static even as the targeting profile changes, as well as highlighting both the benefits and cautions of targeting multiple disparate targets in cells. Unlike our other projects, Paper IV focused on understanding the structure and function of DHODH. We studied a purified DHODH lacking the transmembrane domain using native protein nano-electrospray mass spectrometry (nESI-MS). Firstly, we identified MS conditions that allowed for the DHODH to spray and isolated a high m/z range that corresponded to the molecular weight of the enzyme plus the bound FMN cofactor. Ion mass spectrometry was conducted to differentiate between the holo- and apo- DHODH, with the holo-DHODH corresponding to a compact formation suggesting that folded DHODH with FMN present can be preserved in the gas phase. We next incubated lipids that constitute the human mitochondrial membrane with DHODH and analysed the interaction in the gas phase. Complexes with both PE and CDL were evident, but complexes with PC were not easily detected. The next finding was that an intact protein-cofactor complex was required for the DHODH inhibitor, brequinar, to bind thus confirming that brequinar binding to DHODH is not random, but requires properly structured DHODH. Finally, MD simulations were conducted using both full length and truncated protein associated with a model PE bilayer. These models established that DHODH sits on the surface of the lipid bilayer loosely and is anchored in place by the transmembrane helix and this anchorage holds DHODH in the correct orientation to allow insertion of coenzyme-Q10 into the quinone tunnel of DHODH.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.

Phosphorylation is one the most studied and important post translational modifications. Nano electrospray mass spectrometry coupled with traveling wave (T-Wave)-based ion mobility has been used to filter for phosphorylated peptides in tryptic protein digests. T-Wave parameters have been optimized to maximize the separation between phosphorylated and non-phosphorylated peptides. A method to calibrate the T-Wave device, to provide estimates of collision cross sections, is presented, and these estimates are in excellent agreement with values obtained on drift cell instrumentation. Phosphorylated peptides have smaller cross sections which enables their separation from non-phosphorylated peptides of the same m/z. Post-mobility fragmentation is used to obtain the primary sequence for peptides of interest. This approach is shown to have potential as an additional screen for phosphorylated peptides, where up to 40% of observed peptides can be eliminated from the study.

Ceramides covalently bound to keratinocytes are essential for the barrier function of the skin, which can be disturbed in diseases, such as psoriasis and atopic dermatitis. These ceramides of the classes omega-hydroxyacyl-sphingosine and omega-hydroxyacyl-6-hydroxysphingosine contain an omega-hydroxy fatty acid. For their separation and identification, a new analytical approach based on normal phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry and tandem nano-electrospray mass spectrometry, respectively, is presented here. Tandem mass spectrometry provided structural information about the sphingoid base as well as the fatty acid moieties. The chain lengths of the bases ranged from C12 to C22, the chain lengths of the fatty acids varied between C28 and C36. In total, 67 ceramide species have been identified in human skin. The analytical methods presented in this work can be helpful for investigating alterations in the ceramide composition of the skin as seen in psoriasis, atopic dermatitis, and diseases with impaired epidermal barrier function.

To compare the lipid composition of human meibum across three different meibum expression techniques.Meibum was collected from five healthy non-contact lens wearers (aged 20-35 years) after cleaning the eyelid margin using three meibum expression methods: cotton buds (CB), meibomian gland evaluator (MGE) and meibomian gland forceps (MGF). Meibum was also collected using cotton buds without cleaning the eyelid margin (CBn). Lipids were analyzed by chip-based, nano-electrospray mass spectrometry (ESI-MS). Comparisons were made using linear mixed models.Tandem MS enabled identification and quantification of over 200 lipid species across ten lipid classes. There were significant differences between collection techniques in the relative quantities of polar lipids obtained (P.05). The MGE method returned smaller polar lipid quantities than the CB approaches. No significant differences were found between techniques for nonpolar lipids. No significant differences were found between cleaned and non-cleaned eyelids for polar or nonpolar lipids.Meibum expression technique influences the relative amount of phospholipids in the resulting sample. The highest amounts of phospholipids were detected with the CB approaches and the lowest with the MGE technique. Cleaning the eyelid margin prior to expression was not found to affect the lipid composition of the sample. This may be a consequence of the more forceful expression resulting in cell membrane contamination or higher risk of tear lipid contamination as a result of reflex tearing.

FSH glycosylation macroheterogeneity in pituitary and urinary hFSH samples was evaluated by Western blotting. Microheterogeneity in two highly purified urinary and pituitary hFSH preparations was evaluated by nano-electrospray mass spectrometry of peptide-N-glycanase-released oligosaccharides. An age-related loss of hypo-glycosylated hFSH in individual female pituitaries was indicated by progressively reduced abundance of hFSH21 relative to hFSH24. Urinary hFSH was evaluated as a potentially non-invasive indicator of glycoform abundance, as the only way for pituitary FSH to reach the urine is through the blood. Both highly purified and crude postmenopausal urinary hFSH preparations possessed the same amount of hFSH21 as postmenopausal pituitary gland FSH. Considerable microheterogeneity was encountered in both pituitary and urinary hFSH glycan populations, as 84 pituitary hFSH glycan ions were observed as compared with 68 urinary hFSH glycans. The biggest quantitative differences between the two populations were reduced abundance of bisecting GlcNAc-containing and fucosylated glycans, along with sulfated glycans in the urinary hFSH glycan population. The relative abundance of sialic acid and glycan antenna did not rationalize the retarded electrophoretic mobilities of the urinary hFSHb21- and a-subunit bands relative to the corresponding pituitary hFSH bands, as the most abundant glycans in the former possessed only 2 more branches and the same sialic content as in the latter. Site-specific glycosylation information will probably be necessary.

Degradation of proinsulin C-peptide in mouse kidney and human placenta extracts was studied using reverse-phase high-performance liquid chromatography and nano-electrospray mass spectrometry. In total, 15 proteolytic cleavage sites were identified in human and mouse C-peptides. Early sites included the peptide bonds N-terminal of Val/Leu10, Leu12, Leu21, Leu24 and Leu26 in different combinations for the two tissues and two peptides. Notably, these cleavages were N-terminal of a hydrophobic residue, and all but one N-terminal of Leu. A late degradation product of the human peptide detected in the kidney extract was the C-terminal hexapeptide, containing just one residue more than the biologically active C-terminal pentapeptide of C-peptide. We conclude that the degradation of C-peptide in kidney and placenta follows similar patterns, dominated by endopeptidase cleavages N-terminal of Leu.

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.

Dry eye is a highly prevalent disease causing symptoms of discomfort that are severe and disabling for many sufferers. One of the leading causes of the evaporative form of this complaint is believed to be Meibomian Gland Dysfunction (MGD). Although clinical manifestations of MGD are easily observed, the association between the morphology and function of the meibomian gland and the symptoms experienced by those affected, remains controversial. Better understanding of this relationship may help to predict those at risk and will contribute to development of effective treatments and preventative strategies. This thesis describes a series of studies designed to investigate the relationship between subjective ocular symptomatology and both meibomian gland morphology and function. Grading scales commonly used to assess meibomian gland morphology and lid wiper epitheliopathy were validated and found to offer only moderate sensitivity to detect changes. Objective assessment systems incorporating image analysis software were developed which permitted highly repeatable and accurate assessment of meibomian gland morphology and lid wiper epitheliopathy. The Korb meibomian gland evaluator and the meibomian gland forceps were identified as the optimal meibum expression methods. Tears and meibum were collected and analysed using nano-electrospray, mass spectrometry. These techniques were able to identify and quantify the major classes of component lipids. The lipid profiles derived showed good repeatability, both within and between days. A comparison between tears and meibum showed them to have similar lipid profiles, apart from phospholipids, which occurred roughly four orders of magnitude more abundantly in tears. This confirmed that meibum is not the major source of phospholipids in tears. No clear relationship between meibomian gland morphology and symptoms of discomfort was found, with the possible exception that meibomian gland coverage of the upper lid is reduced in those with more severe symptoms. For meibomian gland function, weak to moderate associations between ocular symptomatology and the position of Marx’s line and the frequency of eye rubbing were observed. Meibum quality and the relative proportion of (O-acyl)-omega-hydroxy fatty acid (OAHFA) in tears were found to potentially discriminate those with the most severe symptoms from the rest of the sample.

The application of capillary electrophoresis/nano-electrospray mass spectrometry to the multi-residue analysis of a large number of sulfonamide antibiotics in milk samples is reported. Tandem mass spectrometric (MS/MS) techniques including precursor ion scans and multiple reaction monitoring were used to identify residues at the low ppb level to the ppt level. Three pairs of isomeric sulfonamides were targeted that differ only in the positions of the nitrogen and oxygen atoms in the heterocyclic aromatic rings of the molecules. Conventional MS/MS analysis yielded no isomer-specific ions. Therefore, a quasi-MS/MS/MS method was applied to overcome these limitations. In-source collision-induced dissociation (CID) was used as a quasi-MS/MS stage to generate ions arising from the heteroaromatic amine moiety. In a second MS/MS step these ions were isolated and made to undergo CID in the collision quadrupole to yield isomer-specific ions. Examples of the application of these methodologies to the analysis of milk extracts are illustrated. © 1997 by John Wiley & Sons, Ltd.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.

The influence of the diameter of the spray tip employed for nano-electrospray mass spectrometry (nano-ES-MS) upon mass spectral charge state distributions was investigated using angiotensin I (M(r) = 1296), insulin (M(r) = 5774), and ubiquitin (M(r) = 8560) as test analytes. Under a variety of experimental conditions, the charge state distributions of the test peptides and protein consistently shifted toward higher values as the tip orifice diameter decreased. This finding indicates that the use of narrow diameter capillaries can promote the formation of higher charge state ions that are more reactive precursors in tandem mass spectrometry experiments. A detailed comparison of charge state distributions obtained for nanospray capillaries of varying diameters was undertaken while systematically varying experimental parameters such as sample flow rate, analyte concentration, solvent composition, and electrospray current. The general tendency to obtain higher charge states from narrow diameter capillaries was conserved throughout, but tips with smaller orifices were more sensitive to sample flow rate (the average charge state was lowered significantly as flow was raised), whereas tips with bigger orifices were more sensitive to analyte concentration and pH of the solution (as each was lowered, the average charge state increased).

Mass spectrometry is an important analytical tool in biological and biochemical research. The speed, accuracy and sensitivity is unmatched by conventional analytical techniques. Identification of proteins and characterisation of their primary structure is a rapidly growing field in the post-genomic era, where matrix-assisted laser desorption/ionisation time-of-flight peptide mass fingerprinting combined with electrospray tandem mass spectrometry is well suited to solve the questions. This thesis deals with the strength and diversity of applying mass spectrometry to biochemical projects. The study covers sample preparation, protein identification, characterisation of post-translational modifications and mechanistic aspects of gas-phase cleavage of protonated peptides. The characteristics of a quadrupole - time-of-flight tandem mass spectrometer, used throughout this study, were investigated in a project where the thyroid hormone receptor ligand binding domain (TR-LBD) was analysed. Carboxymethylation of the TR-LBD in its folded conformation and identification of the alkylated Cys residues allowed estimates of tertiary structure relationships within the protein. In the tryptic digest, the peptides were mass measured with an accuracy of 2 ppm, and product ions generated by tandem mass spectrometry were measured to an accuracy of 20 ppm when internal calibrants were used. Proline-rich proteins (PRPs) are predominant in saliva and nano-electrospray mass spectrometry was employed to identify their degradation products after treatment with Streptococcus and Actinomyces microorganisms. Several of the peptides generated had a Cterminal Pro-Gln bacteria binding sequence, and release of a pentapeptide with innate immunity properties was suggested. In another study it was shown that PRP-1 and its Cterminally truncated form PRP-3 are present in two forms in human saliva, one of which is 0hexuronidated at Ser 17 in the biologically active N-terminal segment. Nano-electrospray tandem mass spectrometry of PRP-1 tryptic peptides was carried out to determine the 0hexuronidation. It was then also found that peptides containing the PQGPPQQGG consensus repeat reveal a facile cleavage of the Gln 2-Gly 3 peptide bond in the gas-phase upon collision-induced dissociation. The data indicate involvement of the G1n side-chain in the fragmentation mechanism. Phosphorylation is a common post-translational modification of proteins but only known in a few prohormone precursors. Using nano-electrospray tandem mass spectrometry, a phosphorylated form of porcine peptide YY was now identified and the modification was localised to Ser 13. In-gel digestion of gel-separated proteins has many advantages in terms of sensitivity and high-throughput for protein identification. However, the coverage of the primary structure by mass spectrometric analysis for detection of post-translational modifications is generally poor. An alternative method was developed that uses electroblotting of the gel-separated proteins to polyvinylidene difluoride membranes followed by protein extraction and digestion in solution. Compared to in-gel digestion, this procedure results in improved sequence coverage (by approximately 30%) and it was applied to identify protein methylation and acetylation sites. DNasel affinity chromatography in combination with nano-electrospray tandem mass spectrometry identified a specific subset of actin-associated heterogeneous nuclear ribonucleoproteins (hnRNP) from rat liver pre-messenger RNP particles. The proteins were identified as being a novel hnRNP A2 isoform, three hnRNP A3 forms and a novel A/B type of hnRNP designated DBP-40. DBP-40 binds directly to actin in vivo, and it is also capable of individually binding RNA as well as hnRNP A2 and A3 in the presence of actin.

Phosphorylation of the head domains of intermediate filament proteins by second messenger-dependent kinases is important in regulating filament assembly. In the case of neurofilaments, head domain phosphorylation is known to be important in assembly, but few sites have been identified. Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits. Neurofilament polypeptides were purified from adult rat brain, and fractions containing a mixture of NF-L and NF-M were nonradioisotopically phosphorylated with PKA prior to proteolytic digestion of the polypeptides in situ in polyacrylamide excised from SDS gels. Sites of phosphorylation were determined by mass spectrometric analysis of mixtures enriched in tryptic phosphopeptides. In NF-L, four novel sites were identified: serines 12, 41, and 49 in the head domain and serine 435 in the carboxyl-terminal tail domain, and data consistent with phosphorylation of serine 2 were obtained. Recombinant rat NF-L protein was also phosphorylated with PKA, and the same serines were identified as phosphorylation sites, with two additional sites, serine 43 and probable phosphorylation of serine 55. In NF-M, one novel site, serine 1 in the amino-terminal head domain, was found to be phosphorylated, and serine 46, also in the amino-terminal head domain, was confirmed as a PKA phosphorylation site.

This work investigates in greater detail than in previous studies the effect of geometry on the relationship between emitted flow rate and applied potential difference in cone-jet mode electrospray systems. The magnitude of the flow rate to voltage relationship is demonstrated to be sensitive to numerous geometric parameters. An explanation of this variation is offered; it is demonstrated that in the cone-jet mode of operation the change of flow rate with the applied extraction voltage is due to the change in electric field at the tip of the emitter. By a finite element method simulation of the assumed electrostatic process the analysis is further extended to include all geometric parameters. The results outlined show the change of flow rate with applied voltage in cone-jet mode electrospray can be significant. This dependence will, under some conditions, have a considerable effect on the electrospray flow rate, and consequently current and droplet size. This has implications on electrospray applications involving the use of the applied voltage to extract the sprayed solution, including nano-electrospray mass spectrometry techniques and some forms of electrospinning.