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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.

We present here novel 2D 'nib-like' interfaces for nano-electro spray mass spectrometry (nanoESI-MS) applications. The design, fabrication and testing of three generations of such sources are outlined. The two first generations of nibs were fabricated using a negative photoresist (SU-8); they consisted of prototypes which allowed us to validate the idea of a nanoESI nib tip. They had respectively 20/spl times/30 /spl mu/m and 8-16/spl times/35 /spl mu/m section dimensions at their outlet. First generation nibs were seen not to have appropriate dimensions for nanoESI applications. Testing of the second generation demonstrated the influence of outlet orifice dimensions on the ionization process and on the electrospray performance. A third generation of micro-nibs was fabricated using polycrystalline silicon (poly-Si) and silicon-based surface micromachining techniques. These latter were highly planar and had much smaller tip dimensions of approximately 2/spl times/2 /spl mu/m. MS testing on an ion trap mass spectrometer, with standard peptides initially, and then samples of various natures, showed the feasibility of the 'nib-like' approach and the subsequent improvements obtained as the design and fabrication of the interfaces evolved.

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.

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.