Assisted Laser Desorption Mass Spectrometry Research Articles

Identification of Pseudomonas aeruginosa exopolysaccharide Psl in biofilms using 3D OrbiSIMS.

Secondary ion mass spectrometry (SIMS) offers advantages over both liquid extraction mass spectrometry and matrix assisted laser desorption mass spectrometry in that it provides the direct in situ analysis of molecules and has the potential to preserve the 3D location of an analyte in a sample. Polysaccharides are recognized as challenging analytes in the mass spectrometry of liquids and are also difficult to identify and assign using SIMS. Psl is an exopolysaccharide produced by Pseudomonas aeruginosa, which plays a key role in biofilm formation and maturation. In this Letter, we describe the use of the OrbiTrap analyzer with SIMS (3D OrbiSIMS) for the label-free mass spectrometry of Psl, taking advantage of its high mass resolving power for accurate secondary ion assignment. We study a P. aeruginosa biofilm and compare it with purified Psl to enable the assignment of secondary ions specific to the Psl structure. This resulted in the identification of 17 peaks that could confidently be ascribed to Psl fragments within the biofilm matrix. The complementary approach of the following neutral loss sequences is also shown to identify multiple oligosaccharide fragments without the requirement of a biological reference sample.

Direct Detection of Glycated Human Serum Albumin and Hyperglycosylated IgG3 in Serum, by MALDI-ToF Mass Spectrometry, as a Predictor of COVID-19 Severity.

The prefusion spike protein of SARS-CoV-2 binds advanced glycation end product (AGE)-glycated human serum albumin (HSA) and a higher mass (hyperglycosylated/glycated) immunoglobulin (Ig) G3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress syndrome (ARDS) as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct serum dilution, and disulphide bond reduction, method was developed and applied to plasma samples from SARS-CoV-2 seronegative (n = 30) and seropositive (n = 31) healthcare workers (HCWs) and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for the development of COVID-19 ARDS as a result of SARS-CoV2 infection. Furthermore, rapid direct analysis of serum/plasma samples by MALDI-ToF for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

MALDI mass spectrometry monitoring of cyclodextrin-oligolactide derivatives synthesis

This paper aims to expand the range of matrix assisted laser desorption mass spectrometry analysis (MALDI MS) as a tool to establish the reaction kinetics for a particular case study: the synthesis of β-cyclodextrin-oligolactide (CDLA) derivatives through solution ring opening oligomerization (ROO) of d,l-lactide (LA) initiated by β-cyclodextrin (β-CD). Specifically, MALDI MS was used to estimate the molecular weight averages minute changes during the synthesis for a better understanding of the chemical process. The MALDI MS data was compared with 1H NMR for determining the DL-Lactide conversion rate and an excellent agreement level was established. The synthesis process was further studied by observing the effects of the reaction parameters with a special focus on the solvent influence. Thus, we observed that the ROO performed in dimethylformamide and N-methyl-2-pyrrolidone proceeds faster in comparison with dimethylsulfoxide due to an activation process in the LA ring opening reaction. The activation is produced by the amines resulted from the cleavage of the amide bonds in the presence of the β-CD or CDLA derivatives. The products of the degradation process were identified in the MS spectra and structurally confirmed through MS fragmentation experiments.

A comparative study of the complete lipopolysaccharide structures and biosynthesis loci of Bordetella avium, B. hinzii, and B. trematum

A dozen species of human and animal pathogens have been described to date in the Bordetella genus, with the majority being respiratory tract pathogens. Bordetella avium lipopolysaccharides have been shown to be important virulence factors for this bird pathogen. B. hinzii is closely related to the B. avium species, but has also been isolated from humans. B. trematum is associated to ear and blood infections in humans. Its lipid A structure, the biological active moiety of LPS, was found to be closely related to those of B. avium and B. hinzii. It is important to unveil the subtle structural modifications orchestrated during the LPS biosynthetic pathway to better understand host adaptation. The present data are also important in the context of deciphering the virulence pathways of this important genus containing the major pathogens B. pertussis and B. parapertussis, responsible for whooping cough. We recently reported the isolated lipid A structures of the three presented species, following the previously identified O-chain structures. In the present study, we provide details on the free and O-chain-linked core oligosaccharides which were required to characterize the complete LPS structures. Data are presented here in relation to relevant biosynthesis genes. The present characterization of the three species is well illustrated by Matrix Assisted Laser Desorption Mass Spectrometry experiments, and data were obtained mainly on native LPS molecules for the first time.

Abstract 4647: Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation

Abstract Introduction: Mutations of the isocitrate dehydrogenase (IDH)-1/2 genes frequently occur in certain malignancies, including gliomas. Mutant IDH-1/2 proteins gain a new ability to produce oncometabolite 2-hydroxiglutarate (2HG), which results in a different clinical impact on gliomas in comparison to those with a wild-type IDH. The status of IDH mutations has been determined by immunohistochemistry (IHC) using the IDH1-R132H antibody or through the DNA sequencing of IDH1-R132C/R132S; however, its practical applications are still under discussion, and the significance of the oncometabolite 2HG evaluation is not clear. In this study, we analyzed the serum and tissue levels of 2HG in clinical gliomas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) system. Method: Glioma samples (n = 78, matched pairs of serum and flash frozen tissue) were obtained between October 2007 to January 2015 at the National Cancer Center Hospital, Japan. High performance liquid chromatography (Nexera X2, Shimadzu) and triple quad mass spectrometry (QTRAP4500, AB SCIEX) were used for LC-MS/MS analysis. The two MALDI-MSI systems, quadrupole ion trap time-to-flight mass spectrometry imaging (iMScope, Shimadzu), and high-resolution atmospheric pressure mass spectrometry imaging (AP-SMALDI, TransMIT; Q-Exactive, Thermo Fisher Scientific) were used for analyzing the 2HG tissue distribution after coating with 9-aminoacridine or 2,5-dihydroxybenzoic acid. The IDH mutation was determined by direct sequencing and IHC. Results: The median 2HG concentration in serum was 21.43 ng/mL (range: 9.58 to 62.51) in this study, and no significant correlation was observed between the IDH mutation and the wild type status (mutation, n = 28, median 20.2 ng/mL, range 9.58 to 62.51 vs. wild type, n = 50, median 22.88 ng/mL, range 11.51 to 40.32; Wilcoxon test p = 0.1597). The median of the 2HG tissue concentration in the entire group was 0.16 ng/μg, and a significant difference was seen between the IDH mutant and wild type groups (mutation, median 4.86 ng/μg, range 0.51 to 49.86 vs. wild type, median 0.09 ng/μg, range 0.02 to 0.45; Wilcoxon test p = <0.0001). In the MALDI-MSI analysis, the average 2HG ion intensities were 381.62/mm2 in the IDH mutation and 8.25/mm2 in the wild-type IDH tissues (Wilcoxon test p = 0.016), which showed a positive correlation with the 2HG tissue concentration by LC-MSMS analyses (Spearman ρ = 0.87, p = 0.0003). The MSI results indicate a possible link between the 2HG tissue distributions and the histology of IDH mutant sections. Conclusion: Tissue 2HG measurement using mass spectrometric analyses may be a useful alternative in determining IDH mutations. Further research may provide a biological perspective and help gain some insights on the clinical significance of 2HG. Citation Format: Mitsuhiro Hayashi, Hiroaki Aikawa, Makoto Ohno, Koichi Ichimura, Yuko Matsushita, Mayu Ohuchi, Mariko Mizui, Akihiko Yoshida, Yoshitaka Narita, Akinobu Hamada. Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2017-4647

Proteomics goes forensic: Detection and mapping of blood signatures in fingermarks.

A bottom up in situ proteomic method has been developed enabling the mapping of multiple blood signatures on the intact ridges of blood fingermarks by Matrix Assisted Laser Desorption Mass Spectrometry Imaging (MALDI-MSI). This method, at a proof of concept stage, builds upon recently published work demonstrating the opportunity to profile and identify multiple blood signatures in bloodstains via a bottom up proteomic approach. The present protocol addresses the limitation of the previously developed profiling method with respect to destructivity; destructivity should be avoided for evidence such as blood fingermarks, where the ridge detail must be preserved in order to provide the associative link between the biometric information and the events of bloodshed. Using a blood mark reference model, trypsin concentration and spraying conditions have been optimised within the technical constraints of the depositor eventually employed; the application of MALDI-MSI and Ion Mobility MS have enabled the detection, confirmation and visualisation of blood signatures directly onto the ridge pattern. These results are to be considered a first insight into a method eventually informing investigations (and judicial debates) of violent crimes in which the reliable and non-destructive detection and mapping of blood in fingermarks is paramount to reconstruct the events of bloodshed.

Cigarette Smoke-Induced Alterations in Frontal White Matter Lipid Profiles Demonstrated by MALDI-Imaging Mass Spectrometry: Relevance to Alzheimer's Disease.

Meta-analysis has shown that smokers have significantly increased risks for Alzheimer's disease (AD), and neuroimaging studies showed that smoking alters white matter (WM) structural integrity. Herein, we characterize the effects of cigarette smoke (CS) exposures and withdrawal on WM myelin lipid composition using matrix assisted laser desorption and ionization-imaging mass spectrometry (MALDI-IMS). Young adult male A/J mice were exposed to air (8 weeks; A8), CS (4 or 8 weeks; CS4, CS8), or CS8 followed by 2 weeks recovery (CS8 + R). Frontal lobe WM was examined for indices of lipid and protein oxidation and lipid profile alterations by MALDI-IMS. Lipid ions were identified by MS/MS with the LIPID MAPS prediction tools database. Inter-group comparisons were made using principal component analysis and R-generated heatmap. CS increased lipid and protein adducts such that higher levels were present in CS8 compared with CS4 samples. CS8 + R reversed CS8 effects and normalized the levels of oxidative stress. MALDI-IMS demonstrated striking CS-associated alterations in WM lipid profiles characterized by either reductions or increases in phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine) and sphingolipids (sulfatides), and partial reversal of CS's inhibitory effects with recovery. The heatmap hierarchical dendrogram and PCA distinguished CS exposure, duration, and withdrawal effects on WM lipid profiles. CS-mediated WM degeneration is associated with lipid peroxidation, protein oxidative injury, and alterations in myelin lipid composition, including shifts in phospholipids and sphingolipids needed for membrane integrity, plasticity, and intracellular signaling. Future goals are to delineate WM abnormalities in AD using MALDI-IMS, and couple the findings with MRI-mass spectroscopy to improve in vivo diagnostics and early detection of brain biochemical responses to treatment.

MALDI-MS of Pseudo-Alkaloid Taxanes from Taxus canadensis

Six previously reported taxanes with alkaloid side chains were isolated from the methanol extract of the needles of Taxus canadensis. The structures were established on the basis of 1D and 2D NMR spectroscopic data. The identification of different alkaloid side chains in these taxanes by Matrix Assisted Laser Desorption Mass Spectrometry (MALDI-MS) was discussed.

Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells

An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively.

Hemisynthesis of 13 2,17 3-cyclomesopheophorbide- a-enol

Cyclopheophorbides are often major chlorophyll- a degradation products in biogeochemical situations. Authentic standards are unavailable and facile generation of pure compounds is required. 13 2,17 3-Cyclomesopheophorbide- a-enol (mesoCYCLO) was prepared in moderate yields via a known Dieckmann-like condensation of mesopyropheophorbide- a methyl ester (mpPBID aME). MesoCYCLO was purified by flash chromatography over a polymeric reversed phase (PRP-1™) material, negating the requirement for a crystallization step. Structural verification included ultraviolet-visible spectrometry, high resolution matrix (sulfur/CS 2) assisted laser desorption mass spectrometry, and nuclear magnetic resonance.

Curve Data Classification via Functional Principal Component Analysis

We propose a best predicted curve classification (BPCC) criterion for classifying the curve data. The data are viewed as realizations of a mixture of stochastic processes and each subprocess corresponds to a known class. Under the assumption that all the groups have different mean functions and eigenspaces, an observed curve is classified into the best predicted class by minimizing the distance between the observed and predicted curves via subspace projection among all classes based on the functional principal component analysis (FPCA) model. The BPCC approach accounts for both the means and the modes of variation differentials among classes while other classical functional classification methods consider the differences in mean functions only. Practical performance of the proposed method is demonstrated through simulation studies and a real data example of matrix assisted laser desorption (MALDI) mass spectrometry (MS) data. The proposed method is also compared with other multivariate and functional classification approaches. Overall, the BPCC method outperforms the others when the mean functions and the eigenspaces among classes are significantly distinct. For classifying the MALDI MS data, we found that functional classification methods perform better than multivariate data approaches, and the dimension reduction via FPCA is advantageous to improving the accuracy of classification.

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A(16)). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.

Comparison of two ESI-MS based H/D exchange methods for extracting protein folding energies

In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate Δ G H 2 O and m GdHCl values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained Δ G H 2 O and m GdHCl values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear extrapolation method (LEM) plots is not a reliable indicator of the validity of the data obtained using the LEM approach.

Characterization of purified His-tagged CP47-containing photosystem II complexes from a cyanobacterium, Synechocystis sp. PCC 6803

For the determination of the exact structure of PS II complexes, all the polypeptide constituents must be identified. For this purpose, highly active photosystem II (PS II) complexes were purified from a HT-3 mutant of Synechocystis sp. PCC 6803 whose CP47 has been His-tagged. Fluorescence measurements showed the intactness of the donor and acceptor sides of purified PS II complexes. It retained 80% of the initial activity of oxygen evolution even after incubation of the complexes for 2 weeks at 4°C in the dark. Using these highly purified PS II complexes, the polypeptide composition was analyzed by SDS-PAGE which can resolve polypeptides of low molecular weight. Separated polypeptides of the purified PS II complexes were subjected to N-terminal amino acid sequencing and matrix assisted laser desorption (MALDI) mass spectrometry. More than 20 Coomassie-stainable polypeptides were identified. All the polypeptides which was known as PS II components were found, except PsbN. Besides 19 polypeptides of well-known PS II member, some polypeptides, which had not been shown to bind to PS II complexes until now, were found. Among such polypeptides, the sll1638 gene product was found to be highly homologous to PsbQ. These PS II complexes contained the FtsH protein, which was assigned to be a protease of the D1 protein, and the Ycf9 (Orf62 in tobacco), which was suggested to be present in stromal thylakoids. The results obtained here showed the usefulness of this HT-3 strain for the investigation of the structure, function and biogenesis of PS II complexes.

Preparation, Characterization, and In Vitro Evaluation of 1- and 4-Month Controlled Release Orntide PLA and PLGA Microspheres

Purpose. To prepare, characterize and evaluate in vitro sustained delivery formulations for a novel LHRH antagonist, Orntide acetate, using biodegradable microspheres (ms). Methods. Poly(d,l-lactide) (PLA) and poly(d,l-lactide-co-glycolide) (PLGA) were characterized for molecular weight (Mw, Mn) using gel permeation chromatography (GPC) and content of free end carboxyl groups (acid number, AN) by a titration method. 1- and 4-month Orntide ms were prepared by a dispersion / solvent extraction / evaporation process and characterized for drug content (HPLC), bulk density (tapping method), particle size (laser diffraction method), surface morphology (scanning electron microscopy, SEM), and structural integrity of encapsulated peptide by Fourier Transform Matrix Assisted Laser Desorption mass spectrometry (FT-MALDI). Peptide binding to PLA and PLGA and non-specific adsorption to blank ms was studied in 0.1 M phosphate buffer pH 7.4 (PB) and 0.1 M acetate buffer pH 4.0 (AB). In vitro release of peptide was assessed in PB and AB. Results. Mw for the PLGA copolymers varied from 10,777 to 31,281 Da and was 9,489 Da for PLA. AN was between 4.60 and 15.1 for the hydrophilic resomers and 0.72 for the hydrophobic 50:50 PLGA copolymer. Spherical ms (3.9 μ to 14 μ in diameter) with mostly non-porous surface and varying degree of internal porosity were prepared. FT-MALDI mass spectra of the extracted peptide showed that the encapsulation process did not alter its chemical structure. Peptide binding to PLGA and PLA and non-specific adsorption to blank PLGA ms were dependent upon pH and were markedly higher in PB than in AB. The initial in vitro release in PB varied from 0.5 to 26%/24 h but due to substantial binding of the peptide to the polymeric matrix the long-term release in PB could not be determined. Application of a dialysis method allowed for a more accurate determination of in vitro release and a good total drug recovery. Conclusions. Orntide acetate was successfully incorporated into PLA and PLGA ms and the 1- and 4-month in vitro release profiles were achieved by polymer selection and optimization of the manufacturing parameters.

Synthesis and enzymatic stability of oligonucleotides consisting of isonucleosides

Abstract

Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley

The gene encoding the starch debranching enzyme limit dextrinase, LD, from barley ( Hordeum vulgare), was isolated from a genomic phage library using a barley cDNA clone as probe. The gene encodes a protein of 904 amino acid residues with a calculated molecular mass of 98.6 kDa. This is in agreement with a value of 105 kDa estimated by SDS–PAGE. The coding sequence is interrupted by 26 introns varying in length from 93 bp to 825 bp. The 27 exons vary in length from 53 bp to 197 bp. Southern blot analysis shows that the limit dextrinase gene is present as a single copy in the barley genome. Gene expression is high during germination and the steady state transcription level reaches a maximum at day 5 of germination. The deduced amino acid sequence corresponds to the protein sequence of limit dextrinase purified from germinating malt, as determined by automated N-terminal sequencing of tryptic fragments coupled with matrix assisted laser desorption mass spectrometry. The sequenced peptide fragments cover 70% of the entire protein sequence, which shows 62% and 77% identity to that of starch debranching enzymes from spinach and rice and 37% identity to Klebsiella pullulanase. Sequence alignment supports the multidomain architecture and identifies both secondary structure elements of the catalytic (β/α) 8-barrel substrate, catalytic residues, and specificity associated motifs characteristic of members of the glycoside hydrolase family 13 which cleave α-1,6-glucosidic bonds. A remarkable distribution of the secondary structure elements to individual exons is observed.

Fractionation of a wood tar pitch by planar chromatography for the characterisation of large molecular mass materials

A commercial tar pitch derived from pine wood – Massen Pine ( Pinus Massonia) and sold as Stockholm tar has been fractionated by planar chromatography with examination of the fractions by size exclusion chromatography in NMP eluent, by UV-fluorescence and by matrix assisted laser desorption mass spectrometry. The relatively small molecules, mobile in planar chromatography, are shown to be non-polar. Large molecules were found in each fraction, corresponding in SEC elution times up to polystyrenes of molecular mass of at least 1.8 million. Size exclusion chromatography profiles by UV light absorbance showed differences in relative absorbance of different wavelengths for large and small molecules, implying differences in structures. MALDI mass spectra indicated molecules of mass of several thousand mass units with the upper limit of mass not defined. Planar chromatography provides a fast, cheap method of isolating large molecular mass fractions of this biomass tar.

Absolute calibration of size exclusion chromatography for coal derivatives through MALDI-m.s.

This paper reports the fractionation and subsequent examination of two, very different coal derived materials, a coal tar pitch and a commercial liquefaction extract. These were separated on the basis of their retention in a typical preparative size exclusion chromatography apparatus using THF as the eluent. Matrix assisted laser desorption mass spectrometry, using the small molecules of the sample as matrix, has been used to examine the various fractions from preparative s.e.c. and the peak mass intensities have been used to define an absolute calibration of the s.e.c. separation. Limitations of the methods are discussed. The u.v.-fluorescence spectra of the fractions were recorded and trends in the spectra are described. The fractions were also analyzed by size exclusion chromatography employing N-methyl-2-pyrrolidinone as the chromatographic eluent. The main advantages of this system over traditional solvents is the avoidance of surface adsorption effects observed with THF and the more complete solution of coal-derived material.

Lipocortin 1 co-associates with cytokeratins 8 and 18 in A549 cells via the N-terminal domain

An affinity chromatography strategy was used to search for proteins in A549 cells which interact with the N-terminus of lipocortin 1 (annexin 1). Using the biologically active fragment Lc13–25 as the affinity ligand, two proteins of molecular weight (m.w.) 52 and 48 kDa were extracted. Affinity blots of these proteins bound iodinated Lc13–25. Partial tryptic digests of these proteins were analysed by matrix assisted laser desorption mass spectrometry and found to display fragmentation patterns with a strong similarity to those of cytokeratin 8 and 18 respectively. Subsequent blotting with a panel of specific cytokeratin antibodies strongly supported the idea that the two proteins were cytokeratin 8 and cytokeratin 18. Cytokeratin 8 was isolated from A549 cells in intermediate filament (IF) preparations which were also found to contain lipocortin 1 as a potential intermediate filament associated protein (IFAP). This association persisted throughout cycles of IF assembly and disassembly. Dual-labelling immuno-histochemistry in A549 cells showed strong co-localization of lipocortin 1 and cytokeratin 8. The implications of this finding are discussed in the light of the biological activity and possible function of lipocortin 1.