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Dimeric M315 is transported into mouse and rat milk in a degraded form.

The controversial issue of serum to milk transport of IgA in rodents was addressed in experiments that evaluated the molecular integrity and antigen-binding ability of the dimeric IgA (dIgA) recovered in the stomachs of rat and mouse pups suckling dams which had been administered homogeneous, dimeric M315 intravenously (i.v.). Four rat and three mice dams were i.v. administered affinity-purified, 125I-dIgA anti-DNP (M315) of which greater than 83% could bind DNP after iodination, and of which greater than 82% could be captured by solid phase anti-M315. Dams were given the labelled dIgA either 1-day or 8-days postpartum. Twenty-three hours after i.v. administration, the radioactivity of each pup, its stomach contents and an aliquot of serum were analyzed for total radioactivity, anti-DNP activity and for the molecular integrity of the recovered I-125. Data showed that 11-43% of 125I-activity given to the dam could be recovered from the stomach contents and sera of the pups after this time. Immunoassay revealed that less than 2% of the recovered radioactivity could bind DNP, i.e. a loss of 98% of functional antibody. It was calculated from ultracentrifugational analyses that less than 0.7% of the 125I-dIgA had been transported intact to the suckling neonates. Analyses of stomach milk and neonatal sera by sucrose density gradient ultracentrifugation revealed that almost all recovered radioactivity was in the form of low mol. wt fragments. Data fail to support the concept of an active mechanism for the transport of intact IgA from serum to milk in rodents during early or mid-lactation.

Animals↗

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes.

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.

Animals↗

Recovery of skeletal muscle after laceration and repair.

Few data are available regarding structural and functional recovery of lacerated skeletal muscle after repair. This study used the extensor digitorum longus muscle of rabbits to document recovery after laceration and repair and a healing period of 12 weeks. Completely lacerated muscles recovered approximately 50% of their ability to produce tension but were able to shorten to 80% of normal. Partially lacerated muscle bellies recovered approximately 60% of their ability to produce tension and normal ability to shorten. Histologic specimens revealed that the lacerations did not cause necrosis of either segment. After the recovery period, histologic and histochemical sections revealed that the distal segment initially isolated from the nerve supply showed histologic changes of fiber atrophy and size variability, increased fibrosis, and nuclear centralization suggestive of denervation. Dense scar separated the two fragments. Data show that skeletal muscle can recover useful but not normal function after laceration and repair.

Animals↗

Accelerated on-column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis.

The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4,5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4,5-dihydro-1H-imidazole. Complete tagging of the peptides by on-column reaction could be obtained when using D2O, as compared to water-based reaction. Methylation of cysteine residues was enhanced when beta-mercaptoethanol was added in the reactant solution.

Amino Acid Sequence↗

Structural characterization of proteins and peptides.

The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. Proteins of interest are typically available in low microgram amounts or even less. The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Structural analysis can be done by Edman degradation or mass spectrometry (MS). Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2-5 pmol of a purified peptide. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. It is also the method of choice for sequencing unknown proteins/ peptides and modified peptides. MS has routinely been used with peptides in the range of 100 fmol or even less. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS is a very fast method that can be automated. It is the method of choice for sensitive analysis and large-scale applications (proteomics). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ionization). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). This approach requires that the protein is known and listed in the database. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.

Chromatography, High Pressure Liquid↗

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker.

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.

DNA↗

Methanogen communities along a primary succession transect of mire ecosystems.

Peat accumulating mires are important sources of the greenhouse gas methane. Methane emissions and methanogenic Archaea communities have been shown to differ between fens and bogs, implying that mire succession includes an ecological succession in methanogen communities. We investigated methane production and the methanogen communities along a chronosequence of mires (ca. 100-2,500 years), which consisted of five sites (1-5) located on the land-uplift coast of the Gulf of Bothnia. Methane production was measured in a laboratory incubation experiment. Methanogen communities were determined by amplification of a methyl coenzyme M-reductase (mcr) gene marker and analyzed by terminal-restriction fragment length polymorphism. The terminal-restriction fragment length polymorphism fingerprinting resulted in 15 terminal restriction fragments. The ordination configuration of the terminal restriction fragments data, using nonmetric multidimensional scaling, showed a clear gradient in the methanogen community structure along the mire chronosequence. In addition, fingerprint patterns of samples from the water table level and 40 cm below differed from one another in the bog site (site 5). Methane production was negligible in the three youngest fen sites (sites 1-3) and showed the highest rates in the oligotrophic fen site (site 4). Successful PCR amplification using mcr gene primers revealed the presence of a methanogen community in all five sites along the study transect.

Biodiversity↗

[An inhibitor of enteropeptidases and trypsin from the bovine duodenum].

Enteropeptidase inhibitor (DI) was isolated from bovine duodenum during purification of this enzyme. DI was purified by affinity chromatography on immobilised trypsin. DI preparations contain two main components: DI-9 (9 kD) and DI-20 (20 kD). The N-terminal amino acid sequence 1-19 of DI-9 is highly homologous to the Kunitz inhibitor (BPI). Molecular weights of DI-9 and BPI are the same (gel electrophoresis data). Fragment 1-19 of DI-9 differs from the corresponding region of BPI only at the position 17: DI-9 contains Ala-17 instead of Arg in BPI. The homology of N-terminal amino acid sequence 1-25 of DI-20 with the corresponding regions of some phospholipases A2 suggests that this protein is a new intestinal phospholipase A2. Inhibitor DI-9 and phospholipase DI-20 are probably isolated in a common lipoprotein complex. The only earlier known in vitro inhibitor of enteropeptidase, BPI, was localised in vivo in different tissues with this enzyme. In our opinion the Kunitz-type inhibitor DI-9 is, a physiological inhibitor of enteropeptidase.

Amino Acid Sequence↗

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry.

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.

Chromatography, Liquid↗

Alternative base-calling algorithm for DNA sequencing based on four-label multicolor detection.

A simple base-calling scheme based on four-label multicolor detection is suggested for DNA sequencing. The entire spectra of the dye labels were used for identification. Specifically, the maxima of the emission spectra rather than the intensity ratios at selected wavelengths are used to provide excellent discrimination. Capillary gel electrophoresis was used for the separation of DNA fragments. Data acquisition and analysis compatible with fast and high-throughput imaging detection was accomplished. The accuracy of base calling of PGEM/U DNA from the raw data obtained with 5 nm and 7 nm spectroscopic resolution were 98.4% for 386 bases and 98.4% for 385 bases. Base calling of M13mp18 DNA showed 98.3% accuracy for 420 bases.

Algorithms↗

Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database.

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.

Colonic Neoplasms↗

Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry.

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.

2-Propanol↗

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry.

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.

Amino Acid Sequence↗

Metabolism studies of the anti-tumor agent maytansine and its analog ansamitocin P-3 using liquid chromatography/tandem mass spectrometry.

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.

Animals↗

Determination of 12 pueraria components by high-performance liquid chromatography-mass spectrometry.

Puerariae radix, a commonly used Chinese herb drug derived from the dried root of legume plant, contains a series of isoflavones as its chief pharmacologically active constituents. Using 12 pueraria components as markers, an LC-UV-MS method requiring less than 60 min, was developed for estimating the quality of pueraria samples within 60 min. Extracts were analyzed using a Cosmosil 5C18-MS column, by gradient elution with an aqueous solution of acetic acid and methanol-ACN at a flow-rate of 1.0 mL/min. Peaks were detected at 254 nm and each peak was identified by LC/MS. The reproducibilities (RSD) of this proposed method, on the basis of peak-area ratios from six replicate injections, were 0.93-1.42% (intraday) and 1.05-1.63% (interday) at a detection limit of 0.12-0.78 microg/mL. Most of the markers used in this study can be classified, respectively, into three major categories, namely, isoflavones, O-glycosidic isoflavones, and C-glycosidic isoflavones. The structures of the compounds were determined from LC-MS fragmentation data and data from the literature.

Acetic Acid↗

Identification of glycoconjugates in the urine of a patient with congenital disorder of glycosylation by high-resolution mass spectrometry.

More than 150 molecular species were detected in a single glycoconjugate fraction obtained from urine of a congenital disorders of glycosylation (CDG) patient by use of high-resolution FT-ICR MS. With respect to its high-mass accuracy and resolving power, FT-ICR MS represents an ideal tool for analysis of single components in complex glycoconjugate mixtures obtained from body fluids. The presence of overlapping nearly isobaric ionic species in glycoconjugate mixtures obtained from CDG patient's urine was postulated from fragmentation data of several precursor ions obtained by nanoESI Q-TOF CID. Their existence was confirmed by high-resolution/high-mass accuracy FT-ICR MS detection. High-resolution FT-ICR mass spectra can, therefore, be generally considered for glycoscreening of complex mixture samples in a single stage. From the accurate molecular ion mass determinations the composition of glycoconjugate species can be identified. Particular enhancement of identification is offered by computer-assisted calculations in combination with monosaccharide building block analysis, which can be extended by considerations of non-carbohydrate modifications, such as amino acids, phosphates and sulfates. Taking advantage of this strategy, the number of compositions assigned to mass peaks was significantly increased in a fraction obtained from urine by size exclusion and anion exchange chromatography.

Congenital Disorders of Glycosylation↗

Improving the reliability and throughput of mass spectrometry-based proteomics by spectrum quality filtering.

In contemporary peptide-centric or non-gel proteome studies, vast amounts of peptide fragmentation data are generated of which only a small part leads to peptide or protein identification. This motivates the development and use of a filtering algorithm that removes spectra that contribute little to protein identification. Removal of unidentifiable spectra reduced both the amount of computational and human time spent on analyzing spectra as well as the chances of obtaining false identifications. Thorough testing on various proteome datasets from different instruments showed that the best suggested machine-learning classifier is, on average, able to recognize half of the unidentified spectra as bad spectra. Further analyses showed that several unidentified spectra classified as good were derived from peptides carrying unanticipated amino acid modifications or contained sequence tags that allowed peptide identification using homology searches. The implementation of the classifiers is available under the GNU General Public License at http://www.bioinfo.no/software/spectrumquality.

Adult↗

The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions.

Peptaibols and related peptide antibiotics (peptaibiotics) display diagnostically useful fragmentation patterns during mass spectrometry (FAB-MS, ESI-CID-MS/MS and CID-MSn]. The paper compiles fragmentation data of pseudo-molecular ions reported in the literature as a guide to the rational identification of recurrently isolated and new peptaibols and peptaibiotics. Taxonomic and ecological aspects of microorganisms producing peptaibols and peptaibiotics are discussed.

Amino Acids↗