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PubMed2000

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Amino Acid Sequence↗

PubMed1991

Human immunodeficiency virus-associated Hodgkin's disease. Clinicopathologic studies of 24 cases and preponderance of mixed cellularity type characterized by the occurrence of fibrohistiocytoid stromal cells.

Hodgkin's disease (HD) was diagnosed in 24 patients who were either seropositive for human immunodeficiency virus (HIV) (21) or members of a high-risk group (three), but had not developed acquired immune deficiency syndrome (AIDS). Clinical presentation of the disease was characterized by constitutional symptoms in all, especially fever (23/24) and disseminated disease (22/24) at diagnosis. Mediastinal adenopathy was rare. Bone marrow involvement was particularly frequent (12/24), and a positive bone marrow biopsy preceded lymph node biopsy in 5 of the 12. Histopathologic features of these tumors included an increased number of nonlymphoid stromal cells, i.e., histiocytic and/or fibroblastoid. In some tumors these fibrohistiocytoid stromal cells were arranged in bundles, but distinct nodule with birefringent collagen band formation was not observed. Twenty-two patients were treated, most with combination chemotherapy; one was untreated; one, unknown. Sixteen, including the one untreated, died with disease at 3 to 25 months; one died of an unrelated cause; four were alive at 3 to 24 months; three were lost to follow-up. Frequent bone marrow involvement at presentation suggests the usefulness of the bone marrow biopsy for diagnosis in subjects at risk, especially when they present with spiking fever of unknown origin. Contrary to most previous series, virtually all of our cases were of mixed cellularity type, characterized by increased fibrohistiocytoid stromal cells in place of depleting lymphocytes. The classic nodular sclerosing feature with birefringent collagen band formation was not observed. In conclusion, HIV-associated HD was characterized by advanced stage with fever at presentation, preponderance of mixed cellularity histologic type with increased fibrohistiocytoid stromal cells, and poor outcome. Hodgkin's disease in AIDS patients presents an intriguing biological model to study the role of stromal histiocytes in immunodeficient patients.

Adult↗

PubMed2000

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Alcohol Dehydrogenase↗

PubMed2000

Two-dimensional electrophoresis and mass spectrometry identification of proteins bound by a murine monoclonal anti-cardiolipin antibody: a powerful technique to characterize the cross-reactivity of a single autoantibody.

Antigenic cross-reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross-reactivity of SLE-related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody-secreting B-lymphocytes. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW x BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp-2 cells as assessed by indirect immunofluorescence assay. Proteins from HL-60 extract were separated by 1-D and 2-D PAGE. Western blotting with mAb 4B7 after SDS-PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2-D PAGE. After trypsin in-gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) Reflector or quadrupole-time of flight (Q-TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS-A RNP. Thus, these results showed that 2-D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross-reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B-cell tolerance observed in SLE.

Amino Acid Sequence↗

PubMed2001

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis.

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer-solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water-soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi-angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on-line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.

Electrophoresis, Capillary↗

PubMed2002

Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry.

The separation and characterization of human apolipoproteins and their isoforms was investigated using capillary electrophoresis (CE) in combination with mass spectrometry (MS). The focus of these analyses was the major protein constituents of plasma high-density lipoproteins, apolipoprotein A-I and A-II. Using aqueous buffers in CE, no separation between apolipoprotein A-I and A-II was observed. With the addition of 10-20% acetonitrile, however, the two species could be separated. Furthermore, multiple peaks for each of the apolipoprotein species were observed under these CE conditions. In order to identify and characterize the components, these separations were then coupled with online mass spectrometric detection (CE-MS). Our CE-MS results suggest that the multiple components observed in the acetonitrile-containing CE separation appear to be oxidized forms of the proteins in addition to native forms of the apolipoprotein A-I and A-II. These data are in agreement with previous reports that the methionine residues of the high-density lipoproteins (HDLs) are sensitive to oxidation, which in turn, alters their lipid binding characteristics and secondary structure. In addition to oxidized forms of the proteins, apolipoprotein A-II contained additional components, which varied in mass by 128 Da. The structural differences between these components were determined by proteolytic digestion and tandem MS. Using these techniques, we determined that these components were due to truncation of the C-terminal glutamine amino acid residue on apolipoprotein A-II. These results demonstrate that CE in combination with MS is a promising technique for screening and characterizing isomers of plasma apolipoproteins.

Acetonitriles↗

PubMed1990

Characterization of different microenvironments at the surface of the frog's taste organ.

We used a panel of histochemical techniques to identify and characterize the cell-associated extracellular material at the surface of the frog's taste organ. We employed morphological and histochemical techniques using both the light microscope and the electron microscope. Results show that the apical, external aspect of cells reaching the surface of the taste organ is in close contact with a layer of amorphous material. The histochemical characteristics of this material vary according to the cell type with which it is in contact. Three different microenvironments can be identified at the surface of the frog's taste organ: type 1 microenvironment is associated with the superficial layer of mucus (secretory) cells; type 2 microenvironment characterizes the surface of the so-called wing cells, which reach the surface of the taste organ as thin laminae running among mucus cells; and type 3 microenvironment shrouds the free endings of putative taste cells and is rich in calcium and lipids. Type 2 and type 3 microenvironments fix peroxidase (a sapid macromolecule) with increasing affinity. We conclude that highly differentiated microenvironments exist at the surface of the frog's taste organ, and these could play a role in the chain of biological events leading to the taste sensation. Furthermore, characterization of the cell-associated, specific microenvironments can help clarify the role of the different cell types in the frog's taste organ.

Animals↗

PubMed2005

Microphthalmia with linear skin defects (MLS) syndrome: clinical, cytogenetic, and molecular characterization of 11 cases.

The microphthalmia with linear skin defects (MLS) syndrome (MIM 309801) is a severe and rare developmental disorder, which is inherited as an X-linked dominant trait with male lethality. In the vast majority of patients, this syndrome is associated with terminal deletion of the Xp22.3 region. Thirty-five cases have been described to date in the literature since the first description of the syndrome in the early 1990s. We now report on the clinical, cytogenetic, and molecular characterization of 11 patients, 7 of whom have not been described previously. Seven of these patients have chromosomal abnormalities of the short arm of the X-chromosome, which were characterized and defined by fluorescence in situ hybridization (FISH) analysis. Intriguingly, one of the patients displays an interstitial Xp22.3 deletion, which to the best of our knowledge is the first reported for this condition. Finally we report on the identification and molecular characterization of four cases with clinical features of MLS but apparently normal karyotypes, verified by FISH analysis using genomic clones spanning the MLS minimal critical region, and with genome-wide analysis using a 1 Mb resolution BAC microarray. These patients made it possible to undertake mutation screening of candidate genes and may prove critical for the identification of the gene responsible for this challenging and intriguing genetic disease.

Abnormalities, Multiple↗

PubMed2006

Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of Jacobsen syndrome.

The 11q terminal deletion disorder or Jacobsen syndrome is a contiguous gene disorder. It is characterized by psychomotor retardation, cardiac defects, blood dyscrasias (Paris-Trousseau syndrome) and craniofacial anomalies. We report on a female patient with an approximately 10 Mb interstitial deletion with many of the features of Jacobsen syndrome: A congenital heart defect, dysmorphic features, developmental delay, and Paris-Trousseau syndrome. The karyotype of the patient is 46,XX,del(11)(q24.1q24.3). The interstitial deletion was confirmed using FISH probes for distal 11q, and the breakpoints were characterized by microarray analysis. This is the first molecularly characterized interstitial deletion in a patient with the clinical features of Jacobsen syndrome. The deletion includes FLI-1, but not JAM-3, which will help to determine the critical genes involved in this syndrome.

Abnormalities, Multiple↗

PubMed1997

Characterization of prelymphomatous stages of B cell lymphoproliferation in Sjögren's syndrome.

OBJECTIVE: To determine whether the prelymphomatous stages of B cell lymphoproliferation in Sjögren's syndrome (SS) may be better characterized by the integration of clinical, pathologic, and molecular data, the latter focusing on the expansion, persistence, and dissemination of clonal B cells in the course of the disease. METHODS: Multiple tissue lesions (synchronous from different tissues and metachronous from the same tissue) were evaluated in biopsy specimens obtained from 6 consecutive patients with SS who had an associated lymphoproliferative disorder. Fully benign gastric lesions were evaluated in tissue from an additional 11 patients with SS who had no associated lymphoproliferative disorder. Multiple and complementary molecular analyses of B cell clonality were used: Southern blot, polymerase chain reaction, single-strand conformation polymorphism, DNA sequencing, and hybridization with clonospecific oligoprobes. All the patients were then strictly followed up for the appearance of lymphoma. RESULTS: Different scenarios of SS-associated B cell lymphoproliferation were identified: 1) the ongoing expansion of the same dominant clone, localized or disseminated, in tissue from 2 patients, 1 of whom later developed an overt B cell lymphoma; 2) different dominant clones in different synchronous or metachronous tissues from the remaining 4 patients with an associated lymphoproliferative disorder; and 3) small oligoclonal expansions in 7 of the 11 benign gastric lymphoid infiltrates. CONCLUSION: Prelymphomatous B cell lymphoproliferation in SS was better characterized following integration of the findings. The different types of B cell clonal expansion (oligoclonal or monoclonal, smaller or larger in size, fluctuating or established, localized or disseminated) may imply a different risk of lymphoma progression. An accurate clinical, histopathologic, and molecular characterization may therefore be crucial in future studies aimed at clarifying the pathobiology of SS-associated lymphoproliferation.

Adult↗

PubMed2002

Vibrational, 1H-NMR spectroscopic, and thermal characterization of gladiolus root exudates in relation to Fusarium oxysporum f. sp. gladioli resistance.

Fourier transform Raman (FT Raman) and IR (FTIR) and (1)H-NMR spectroscopies coupled with differential scanning calorimetry (DSC) were applied to the characterization of root exudates from two cultivars of gladiolus (Spic Span and White Prosperity) with different degrees of resistance and susceptibility to Fusarium oxysporum gladioli, the main pathogen of gladiolus. This work was aimed at correlating the composition of root exudates with the varietal resistance to the pathogen. Spectroscopic analysis showed that White Prosperity root exudate differs from Spic Span root exudate by a higher relative amount of the aromatic-phenolic and sugarlike components and a lower relative amount of carbonylic and aliphatic compounds. DSC analysis confirmed the spectroscopic results and showed that White Prosperity root exudate is characterized by an aromatic component that is present in a higher amount than in the Spic Span root exudate. The results are discussed in relation to the spore germination tests showing that White Prosperity, which is characterized by a remarkable resistance toward F. oxysporum gladioli, exudes substances having a negative influence on microconidial germination of the pathogen; root exudates from Spic Span, one of the most susceptible cultivars to F. oxysporum gladioli, proved to have no effect. White Prosperity's ability to inhibit conidial germination of F. oxysporum gladioli can be mainly related to the presence of a higher relative amount of aromatic-phenolic compounds.

Drug Resistance, Fungal↗

PubMed1993

Overcrossing spectra of protein backbones: characterization of three-dimensional molecular shape and global structural homologies.

A procedure is developed and applied to characterize the global shape and folding features of the backbone of a chain molecule. The methodology is based on the following concept: the probability of observing a rigid placement of a backbone in 3-space as a projected curve with N overcrossings. The numerical computation of these probabilities allows one to construct the overcrossing spectrum of a macromolecule at a given configuration. Although the spectrum is built from the knowledge of the nuclear geometry of the main-chain atoms, the shape descriptor overlooks local geometrical features (such as distances and contacts) and provides a characterization of essential (topological) features of the overall fold, such as its compactness and degree of entanglement. In contrast with other shape descriptors, the present approach gives an absolute characterization of the configuration considered, and not one that is relative to an arbitrarily chosen reference structure. Moreover, it is possible to discriminate between folding features that otherwise may not be distinguished when using other geometrical or topological global descriptors. The overcrossing spectrum is proposed as a tool that complements current structural analyses of macromolecules, especially when monitoring structural homologies within a group of related or unrelated polymers. In this work, we apply the methodology to the analysis of proteins having the globin fold. The results are compared with those of other proteins exhibiting similar size and number of residues. Some basic properties of the spectra as a function of the chain length are also discussed.

Macromolecular Substances↗

PubMed2005

Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering.

The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.

Bioreactors↗

PubMed2004

Characterization of amyloidogenic immunoglobulin light chains directly from serum by on-line immunoaffinity isolation.

Primary systemic amyloidosis (AL) is characterized by the overproduction of immunoglobulin light chain proteins by a monoclonal, terminally differentiated B-lymphocyte or plasma cell clone. The free immunoglobulin light chains are deposited in an abnormal conformation as amyloid in a variety of organs in the body. The mechanism of amyloid formation is not well understood, but appears to be associated with some form of cleavage of the immunoglobulin light chain with subsequent aggregate formation. In an attempt to characterize the structure of amyloid-forming light chain proteins we developed an on-line immunoaffinity purification and subsequent characterization of free kappa and free lambda immunoglobulin light chains by electrospray ionization mass spectrometry. The methodology is totally automated and requires 20 micro L of serum. Mass spectral analysis of Bence Jones proteins under non-denaturing conditions was also utilized to examine the tertiary and quaternary structure of light chain proteins and clearly shows covalent dimer formation of lambda type light chain. This type of on-line assay may prove helpful in elucidating distinguishing features capable of discriminating AL from benign monoclonal gammopathies of undetermined significance as well as diagnosing AL.

Amyloid↗

PubMed2006

Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk.

A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.

Animals↗

PubMedDate not supplied

Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein.

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.

Adenomatous Polyposis Coli Protein↗

PubMed1978

Mass spectral characterization of the glucuronide conjugates of terbutryn (2-(t-butylamino)-4-(ethylamino)-6-(methylthio)-s-triazine) metabolites from rats and goat.

Five metabolities from terbutyrn (2-(t-butylamino)-4-(ethylamino)-6-(methylthio)-s-triazine) were characterized to be glucuronide conjugates by interpretation of mass spectral data of their trimethylsilyl derivatives. These were isolated from rat and goat urine. Three of these glucuronides were characterized to be triazinyl-S-glucuronides and two were characterized to be alkyl-O-glucuronides.

Animals↗

PubMed1988

Characterization of polar substituted polycyclic aromatic compounds using high-resolution gas chromatography/mass spectrometry negative ion chemical ionization and positive and negative ion thermospray liquid chromatography/mass spectrometry.

The sensitivity and selectivity of high-resolution gas chromatography/mass spectrometry in the negative ion chemical ionization mode with methane as reagent gas was evaluated for the characterization of polar substituted polycyclic aromatic compounds (PAC). The fragmentation patterns were affected by the nature of the substituent for polar substituted nitro-PAC that showed detection limits of 50 pg at full-scan acquisition. This technique has been applied to the characterization of polar high-performance liquid chromatographic fractions of diesel exhaust particulate (NBS Standard Reference Material 1650) and enabled the identification of 20 PAC of different chemical classes. Among them, hydroxynitro-, dinitro- and nitrosubstituted secondary amines were identified for the first time in diesel exhaust particulate. In addition, 'filament-on' thermospray (TSP) liquid chromatography/mass spectrometry (LC/MS) with positive and negative ions have been used for the characterization of similar polar compounds such as 2-nitroquinoline, 1,8-naphthalic anhydride, naphthalene sulphonic acid and 1,2-hydroxynitronaphthalene. LC analyses were performed on a reversed-phase system using either acetonitrile-water or methanol-water with 0.1 M ammonium acetate and 1% acetic acid as eluent. With negative ion TSP LC/MS a four- to fivefold loss in sensitivity was observed for naphthalene sulphonic acid compared with nitrohydroxy-PAC, that showed a minimum detectable amount of 50 ng in the reconstructed ion chromatogram.

Chromatography, Liquid↗