Specific electrochemical nitration of horse heart myoglobin

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 but was masked in holomyoglobin by the Soret band. In both cases, the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.     

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.     

A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.     

Leucine enkephalin-tyrosinase reaction products — Identification and biological activity

Leucine enkephalin (1 mM) was reacted with mushroom tyrosinase under reductive conditions (ascorbic acid, 50 mM). Reaction products were isolated by high-performance liquid chromatography and identified using electrospray ionization mass spectrometry. The products of the reaction were found to be hydroxylated at the Tyr¹ moiety of the peptide. The major product was a monohydroxylated derivative of leucine enkephalin ([HO-Tyr¹]LE) and the minor product of the reaction was a dihydroxylated derivative ([(HO)₂-Tyr¹]LE). The affinity of [HO-Tyr¹]LE to receptors in rat brain homogenate was compared to that of leucine enkephalin itself. Hydroxylation of LE was found to decrease receptor affinity to both μ and δ opioid receptor sites by a factor of about 20.     

Analysis of sulfatide from rat cerebellum and multiple sclerosis white matter by negative ion electrospray mass spectrometry

The accumulation of sulfatide (sulfatogalactosyl cerebroside) and changes in the sulfatide species present have been examined in the cerebellum of day 6-32 aged rats and in multiple sclerosis (MS) tissue samples. Negative ion electrospray mass spectrometry with daughter and parent ion analyses were used to distinguish the fatty acyl character in the amide linkage of sulfatide; measurement was done by selected ion and multiple reaction monitoring of individually identified sulfatide molecules. Sulfatide accumulation in rat cerebellum shows that 18:0- and hydroxylated 18:0-sulfatide are the first sulfatide molecules detectable. Very long fatty acyl chain sulfatide molecules (>20:0) are present at day 7 and the ratio of non-hydroxylated compared to hydroxylated sulfatide rises as the amount of non-hydroxylated sulfatide increases. 24:1-sulfatide accumulates at a ratio of about 3:1 over 24:0-sulfatide during active myelination. Analyses of the sulfatide in human tissue have shown differences between MS plaque tissues, normal appearing adjacent white matter and control tissues. The findings show that total sulfatide is reduced by 60% in the plaque matter and decreased 25% in adjacent normal appearing white matter. There are significant increases (P=0.05) in the amount of hydroxylation of sulfatide, demonstrated by an increase in the percentage of hydroxylated h24:0-sulfatide (hydroxy-lignoceroyl sulfatide).     

Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.     

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

Isocratic solid phase extraction-liquid chromatography (SPE-LC) interfaced to high-performance tandem mass spectrometry for rapid protein identification

Reversed-phase liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) allows analysis of very complex peptide mixtures at great sensitivity, but it can be very time-consuming, typically using 60 min, or more, per sample analysis. We recently introduced the isocratic solid phase extraction-liquid chromatography (SPE-LC) technology for rapid separation (~8 min) of simple peptide samples. We now extend these studies to demonstrate the potential of SPE-LC separation in combination with a hybrid linear ion trap-Orbitrap tandem mass spectrometer for efficient analysis of peptide samples in proteomics research. The system performance of SPE-LC-MS/MS was evaluated in terms of sensitivity and efficiency for the analysis of tryptic peptide digests obtained from samples consisting of up to 12 standard proteins. The practical utility of the analytical setup was demonstrated by the analysis of <15 microg depleted human serum proteome by a combination of SDS-PAGE and SPE-LC-MS/MS. A total of 88 unique gene products spanning 3 orders of magnitude in serum protein concentration were identified using stringent database search criteria.     

Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry

Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.     

Ion mobility mass spectrometry enables the efficient detection and identification of halogenated natural products from cyanobacteria with minimal sample preparation

Direct observation of halogenated natural products produced by different strains of marine cyanobacteria was accomplished by electrospray ionization and matrix assisted laser desorption ionization and gas phase separation via ion mobility mass spectrometry of extracts as well as intact organisms.     

Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

This paper reviews methods for the analysis of N-linked glycans by mass spectrometry with emphasis on studies conducted at the Oxford Glycobiology Institute. Topics covered are the release of glycans from sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, their purification for analysis by mass spectrometry, methods based on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization for producing fragment ions, and details of their fragmentation. MALDI mass spectrometry provided a rapid method for profiling neutral N-linked glycans as their [M + Na](+) ions which could be fragmented by collision-induced decomposition to give spectra containing both glycosidic and cross-ring fragments. Electrospray ionization mass spectrometry was more versatile in that it was relatively easy to change the type of ion that was formed and, furthermore, unlike MALDI, electrospray did not cause extensive loss of sialic acids from sialylated glycans. Negative ions formed by addition of anions such as chloride and, particularly, nitrate, to the electrospray solvent were stable and enabled singly charged ions to be obtained from larger glycans than was possible in positive ion mode. Fragmentation of negative ions followed specific pathways that defined structural details of the glycans that were difficult to obtain by classical methods such as exoglycosidase digestion.     

Mass spectrometric and chemical stability of the Asp-Pro bond in herpes simplex virus epitope peptides compared with X-Pro bonds of related sequences.

The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography-mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.     

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and isotope ion cluster

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography-electrospray ionization mass spectrometry, gas chromatography-mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS(n) spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.     

Utility of mass spectrometry for proteome ana lysis: part I. Conceptual and experimental approaches

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.     

Protein cross-links: universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry.

A general method of unequivocally identifying and obtaining sequence information on cross-linked peptides derived by proteolytic digestion of cross-linked proteins has been developed. The method is based on isotopic labeling of alpha-amino groups with 2, 4-dinitrofluorobenzene (DNFB) coupled with electrospray ionization mass spectrometry. Proteins containing covalent cross-link(s) are reductively methylated to convert lysine residues to dimethyl lysine. The methylated protein is partially hydrolyzed and the liberated alpha-amino termini are derivatized with an equimolar mixture of DNFB and [(2)H(3)]DNFB. Dinitrophenyl (DNP)-labeled peptides may be fractionated into mono- and bis-DNP pools by chromatography on phenyl media. The bis-DNP peptides are further separated by reverse-phase HPLC and analyzed by electrospray ionization mass spectrometry. The molecular ions of cross-linked peptides are unambiguously identified as 1:2:1 triplets in the mass spectrum resulting from the binomial distribution of isotopic label in the bis-DNP derivative. Sequence information can be elucidated from the unique product ion patterns which are generated from in-source fragmentation at an elevated cone voltage. Analysis of the disulfide cross-linked peptide (VTCG)(2) was undertaken as a proof of concept and the generality of the method was demonstrated by isolating and sequencing the isopeptide bond of polyubiquitin.     

The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus

Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.     

Evidence for two enzymatic pathways for omega-oxidation of docosanoic acid in rat liver microsomes

We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of C22:0 produced not only the hydroxylated product, omega-hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with omega-hydroxy-C22:0 in the presence of either NAD+ or NADPH, C22:0-DCA was formed readily. Formation of C22:0-DCA from either C22:0 or omega-hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD+ as cofactor. Furthermore, omega-oxidation of C22:0 was reduced significantly when molecular oxygen was depleted. The high sensitivity toward the more specific cytochrome P450 inhibitors ketoconazole and 17-octadecynoic acid suggests that hydroxylation of C22:0 and omega-hydroxy-C22:0 may be catalyzed by one or more cytochrome P450 hydroxylases belonging to the CYP4A and/or CYP4F subfamily. This study demonstrates that C22:0 is a substrate for the omega-oxidation system in rat liver microsomes and that the product of the first hydroxylation step, omega-hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD+ or NADPH.     

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.     

内吗啡肽-2的人工合成及其酶促降解

内吗啡肽-2(endomorphin-2)是Zadina等人[1]于1997年发现的一种具有镇痛作用的四肽,它存在于动物和人的中枢神经系统内[2].它是内源性μ阿片受体的激动剂,具有高亲合性(K1=690pmol,L)和选择性(δ/μ=13400、K/μ=7600).