Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.     

Structural analyses of novel glycerophosphorylated alpha-cyclosophorohexadecaoses isolated from X. campestris pv. campestris

Novel periplasmic anionic cyclic glucans produced by Xanthomonas campestris pv. campestris were isolated by trichloroacetic acid treatment and various chromatographic techniques. No report has been made on the presence of substituted cyclic glucans of the Xanthomonas species. We show, for the first time, that X. campestris pv. campestris produces the anionic cyclic glucans with phosphoglycerol residues, the presence of which can be predicted by analyzing the sequence database with the aid of the NCBI RefSeq database. To analyze the structure of isolated anionic cyclic glucans analyses, we used NMR spectroscopy, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOFMS) and electrospray-ionization mass spectrometry (ESIMS). The results suggest that the novel anionic forms of the cyclic glucans of X. campestris pv. campestris are glycerophosphorylated alpha-cyclosophorohexadecaose with one or two phosphoglycerol substituents at the C-6 positions of the glucose residues.     

Metaproteomics Approach To Study the Functionality of the Microbiota in the Human Infant Gastrointestinal Tract

A metaproteomics approach comprising two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) was applied to the largely uncultured infant fecal microbiota for the first time. The fecal microbial metaproteome profiles changed over time, and one protein spot contained a peptide sequence that showed high similarity to those of bifidobacterial transaldolases.     

Metaproteomics approach to study the functionality of the microbiota in the human infant gastrointestinal tract

A metaproteomics approach comprising two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) was applied to the largely uncultured infant fecal microbiota for the first time. The fecal microbial metaproteome profiles changed over time, and one protein spot contained a peptide sequence that showed high similarity to those of bifidobacterial transaldolases.     

Disulfide pairings and secondary structure of porcine beta-microseminoprotein

A sperm motility inhibitor isolated from porcine seminal plasma is identical to porcine beta-microseminoprotein (MSP). Circular dichroism (CD) and nuclear magnetic resonance (NMR) data showed that the native and recombinant porcine MSPs exhibit very similar structure. The five disulfide pairings on porcine MSP were unambiguously assigned based on NMR data and further confirmed using structural calculations. Surprisingly, our derived pairings differ from those recently reported for ostrich MSP based on matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. Furthermore, the secondary structure was determined to comprise one four-stranded and two double-stranded antiparallel beta-sheets. As we know, this is the first detailed secondary structure reported among several types of MSPs.     

In vitro protein complex formation with cytoskeleton-anchoring domain of occludin identified by limited proteolysis

Occludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton. The latter function is accomplished through binding of the C-terminal cytoplasmic region to scaffolding proteins that mediate binding to cytoskeletal actin. We isolated a structural domain from both the bacterial-expressed C-terminal cytoplasmic region of human occludin and native cellular occludin, extracted from epithelial (Madin-Darby canine kidney) or endothelial (human brain) cells, by limited proteolysis with trypsin. This human occludin domain contains the last 119 amino acids as identified by N-terminal sequencing and peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Based on the sequence and secondary structure prediction, this domain contains 4 of 5 alpha-helices in the C-terminal region and is linked to the fourth membrane-spanning region by a loosely structured tethering polypeptide. Comparison of circular dichroism spectra of recombinant proteins corresponding to the entire C-terminal region versus only the binding domain region also supports the interpretation that the helical structural elements are concentrated in that domain. Co-immunoprecipitation of this domain with ZO-2 demonstrated preservation of the specificity of the scaffolding protein-binding function, and binding studies with immobilized ZO-2 suggest the presence of multiple ZO-2 binding sites in this domain. These results provide a basis for development of a structural model of the ZO-binding site that can be used to investigate regulation of tight junction anchoring by intracellular signaling events.     

Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix-assisted laser desorption/ionization mass spectrometry

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.     

Analysis of protein glycosylation by mass spectrometry

We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline beta-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N- and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).     

In Vitro Assembly of [Fe4S4] Cluster in High Potential Iron–Sulfur Protein from Acidithiobacillus ferrooxidans

High-potential iron-sulfur protein (HiPIP) has been proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, which contains an [Fe(4)S(4)] cluster. We report here the assembly of an [Fe(4)S(4)] cluster in HiPIP from A. ferrooxidans ATCC 23270 in vitro in the presence of Fe(2+) and sulfide. The spectra and matrix-assisted laser desorption ionization-time of flight mass spectrometry results of holoHiPIP confirmed that the iron-sulfur cluster was correctly assembled into the protein.     

Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins

Salt bridges between self-associating hen egg white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. The self-assembly of bovine insulin was also investigated using cyanogen. Using high-performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). A method was developed incorporating cyanogen, enzymatic digestion, one-dimensional gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein-protein interfaces. The novelty of this approach is the ease with which cyanogen can be administered to a protein sample and the apparent lack of nonspecific cross-linking side reactions interfering with the analysis.     

Electrochemical investigation of metal ion interactions of a ferrocene deoxyuridine conjugate

The metal ion interactions with ferrocene-modified deoxyuridine (FcdU) have been studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) at a glassy carbon electrode in aqueous medium. Binding constants (K_n+) were determined from voltammetric data, i.e. shifts in potential and changes in limiting current with FcdU and after the addition of different metal ions. Electrospray ionization (ESI) mass spectra and matrix-assisted laser desorption/ionization-time flight mass spectrometry (MALDI-TOF MS) of FcdU recorded in presence of metal ions suggest that FcdU forms stable complexes with Cd²⁺.     

A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode

Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (D-ribose-binding periplasmic protein, D-galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode.     

Isolation,Identification of <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis> AN1 and its Antilisterial Peptide Purification and Characterization

Lactobacillus mucosae strain AN1 isolated from sheep milk and characterized for its probiotic suitability. In vitro evaluation of critical gut endurance properties of this strain were assessed by different screening methods such as bile salt, gastric acid, lysozyme tolerance assays, hemolytic, cholesterol reduction properties, and HT-29 cell line adhesion assay. Antibacterial peptide from this strain was purified using ammonium sulphate precipitation, gel filtration chromatography and reverse-phase HPLC. The molecular mass of peptides was determined by Tricine-SDS-PAGE and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-MS). Purified peptide was named as AN1 having a molecular mass of 10.66 kDa. Helical structures of peptide were determined using circular dichroism spectroscopy. Stability of peptide AN1 towards different parameters such as pH, temperature, organic solvents, proteolytic, and glycolytic enzymes was also analyzed.     

PEGylation of bovine serum albumin using click chemistry for the application as drug carriers

Monomethyl poly(ethylene glycol) (mPEG)-modified bovine serum albumin (BSA) conjugates (BSA-mPEG) were obtained by the mild Cu(I)-mediated cycloaddition reaction of azided BSA (BSA-N(3) ) and alkyne-terminated mPEG. The structure and characteristics of BSA-mPEG conjugates were thoroughly investigated. There were about two PEG chains conjugated onto each BSA molecule as determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. The intrinsic nonspecific binding ability of BSA was used for adsorption and sustained release of both rifampicn and 5-fluorouracil (5-FU). The helical structures of BSA were preserved to a large extent after modification and drug adsorption on BSA was confirmed via circular dichroism spectroscopy. Drugs adsorbed onto the conjugated formulation to a lesser extent than on BSA due to mPEG modification. The in vitro release of both rifampicin and 5-FU, however, indicated that BSA-mPEG can function as a drug carrier. Overall, the click reaction provided a convenient tool for the pegylation of BSA. The biological activity of the BSA-mPEG conjugates, including the drug transportation capacity and biocompatibility, were largely retained.     

Determination of the post-translational modifications of salivary acidic proline-rich proteins

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.     

Proteome analysis by bio-active ceramic water in rat liver: contribution to antioxidant enzyme expression, SOD I

The purpose of this study was to investigate the protective effect of bio-active ceramic water on rat liver. Male Wistar rats were divided into 4 groups of 15 animals each. Groups 1 and 2 were fed bio-active ceramic water and tap water for 4 months, respectively. Groups 3 and 4 were treated with the same condition for 12 months. The changes of protein expression of these four groups were investigated using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven proteins were significantly up-regulated in bio-active ceramic water treated rat liver including aldehyde dehydrogenase I and II, albumin, fructose-1,6-bisphosphatase, and superoxide dismutase I (SOD I). The most highly expressed protein, SOD I with up-regulated enzyme activity, was confirmed by immunoblots as a major antioxidant capable of detoxifying normally generated reactive oxygen species. These data suggest that modified protein expression of the liver contributes to enhance liver function.     

Identification of a Protein That Inactivates the Competence-Stimulating Peptide of Streptococcus pneumoniae

Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.     

The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus

To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.     

Massspectrometric analyses of transmembrane proteins in human erythrocyte membrane

It is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electrospray ionization (LC/ESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and to 2,4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.