Temporal variations of coagulation factor VII activity in mice are influenced by lighting regime

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.     

Mass spectrometric analysis of histone posttranslational modifications

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris

Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.     

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.     

Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.     

Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients

The appearance of asparaginase-resistant acute lymphoblastic leukemia (ALL) in transformed cell lines has been correlated with increased expression of asparagine synthetase (ASNS). Recent measurements using mRNA-based assays have raised doubts, however, as to the importance of ASNS protein in the cellular mechanisms that confer drug resistance upon the leukemic cells. Studies aimed at determining the concentration of ASNS protein in human leukemias are therefore needed to resolve this issue. A mass spectrometry (MS)-based procedure is presented for the direct quantification of ASNS protein concentration in complex sample mixtures. This assay is able to distinguish samples from transformed cell lines that express ASNS over a wide dynamic range of concentration. Importantly, this method directly detects ASNS protein, the functional entity that may be synthesizing sufficient asparagine to render leukemia cells resistant to asparaginase-treatment. We also report the successful use of this MS method, which has lower limits of detection and quantification of 30 and 100 attomoles, respectively, for the first direct measurements of ASNS protein concentrations in four patient blast samples.     

人凝血酶原复合物生产过程中凝血因子活化分析

目的通过比较以组分Ⅲ沉淀和血浆为原料制备人凝血酶原复合物(Prothrombin complex concentrates,PCC)过程中凝血因子活化情况,为选择最适PCC制备原料提供数据支持。方法分别对以组分Ⅲ沉淀和血浆为原料制备PCC过程中中间品的活化的凝血因子活性和人凝血酶活性两个项目进行检定,分析凝血因子的活化情况。观察以组分Ⅲ沉淀为原料制备PCC过程中添加肝素能否抑制PCC中凝血因子的活化。结果以组分Ⅲ沉淀为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均不合格。以组分Ⅲ沉淀为原料制备PCC生产过程中添加肝素后,PCC中间品的活化的凝血因子活性和人凝血酶活性均不合格。以血浆为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均合格。结论组分Ⅲ沉淀为原料制备PCC会增加凝血因子活化的风险,新鲜冰冻血浆可作为制备PCC的原料。     

Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides

Given the extensive efforts applied toward proteomics and research in biomarkers, methods for the simultaneous measurement of proteins, peptides, metabolic intermediates, hormones, etc. in a complex sample may be required in the foreseeable future. Assays based on mass spectrometric detection may be suitable for meeting the demands of such complex samples with sensitivity and specificity. An analytical method for the quantitation of C-reactive protein (CRP), a well-known marker of inflammation, is described. Exact quantities of two synthetic (13)C-labeled CRP tryptic peptides were added as internal standards directly to the sample prior to chemical treatment, trypsinization, and liquid chromatography/mass spectrometry quantitation. C-reactive protein levels based on isotopic response ratios were measured. Intact C-reactive protein was spiked into blank rat urine for chemical and enzymatic treatment, producing linear response ratios of labeled to unlabeled peptides. For rigorous quantitation, standard curves, and quality control samples were prepared in rat urine with highly purified labeled and unlabeled peptides over the 50 pg-5 ng/muL concentration range. Using the same chemical and enzymatic treatment used for digestion of intact CRP, data from these samples demonstrated excellent analytical performance. The method was successfully applied toward the quantitation of urinary C-reactive protein from a study of drug-induced nephrotoxicity.     

Temporal Variations of Coagulation Factor VII Activity in Mice Are Influenced by Lighting Regime

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.     

Purification of human factor VII utilizing O-(diethylaminoethyl)-Sephadex and Sulfopropyl-Sephadex chromatography

A simple procedure for the large scale purification of unactivated human factor VII is described. The initial steps, common to prior purification methods, include adsorption onto barium citrate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. Factor VII is isolated in pure unactivated form by one additional step, Sulfopropyl-Sephadex chromatography. Ten liters of plasma yields 1.3 mg of protein representing approximately 30% recovery.     

Random analysis of amino acid pairs sensitive to variants in human coagulation factor IX precursor

In this study, we used a random approach to determine which amino acid pairs in human coagulation factor IX precursor are more sensitive to its 99 variants. The results show that the randomly unpredictable amino acid pairs are more sensitive to variants.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtii

A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.     

Binding of recombinant human coagulation factor VIII to lipid nanotubes

Cryo-electron microscopy has the power to visualise lipid membranes at the closest to in vivo conditions. The structure of the lipid bilayer can be well resolved and the interactions between lipid-protein and protein-protein molecules followed at the molecular level. We undertook an extended Cryo-electron microscopy study to follow the factor VIII binding to phosphatidylserine containing lipid nanotubes at different lipid composition. Obtaining well ordered tubes is required to define the factor VIII membrane-bound structure. The observed alterations in the arrangement of the protein molecules are indicative for the flexibility of the membrane-bound factor VIII. Understanding the significance of these conformational changes is essential to comprehend the function of factor VIII in coagulation and as a drug for Hemophilia A.     

Mass spectrometric analysis of the reactions catalyzed by -2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of -2-haloalkanoic acids to produce the corresponding -2-hydroxyalkanoic acids. Asp10 of -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.     

Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography--electrospray mass spectrometry

Glycosylation is an important posttranslational modificationin proteins, and aberrant glycosylation occurs in malignancies.Human chorionic gonadotropin (hCG) is a glycoprotein hormoneproduced in high concentrations during pregnancy. It is alsoexpressed as particular glycoforms by certain malignancies.These glycoforms, which are called "hyperglycosylated" hCG (hCGh),have been reported to contain more complex glycan moieties.We have analyzed tryptic glycopeptides of the ß-subunitof hCG of various origins by liquid chromatography (LC) connectedto an electrospray mass spectrometer. Site-specific glycan structureswere visualized by the use of differential expression analysissoftware. hCGß was purified from urine of two patientswith testicular cancer, one with choriocarcinoma, one with aninvasive mole, two pregnant women at early and late gestation,from a pharmaceutical preparation and culture medium of a choriocarcinomacell line. N-glycans at Asn-13 and Asn-30 as well as O-glycansat Ser-121, Ser-127, Ser-132, and Ser-138 were characterized.In all samples, the major type of N-glycan was a biantennarycomplex-type structure, but triantennary structures linked toAsn-30 as well as fucosylation of the Asn-13-bound glycan areincreased in cancer-derived hCGß. There were significantsite-specific differences in the O-glycans, with constant core-2glycans at Ser-121, core-1 glycans at Ser-138, and putativesites unoccupied by any glycan. Core-2 glycans at either Ser-127or Ser-132 were enriched in cancer. The glycans of free hCGßwere larger and had a higher fucose content of Asn-13-linkedoligosaccharides than intact hCG. This may facilitate the detectionof this malignancy-associated variant by a lectin assay. Analysisof hCGh affinity purified with antibody B152 confirmed thatthis antibody recognizes a core-2 glycan on Ser-132.     

Mass spectrometric analysis of protein histidine phosphorylation

Summary. Protein histidine phosphorylation is now recognized as an important form of post-translational modification. The acid-lability of phosphohistidine has meant that this phosphorylation has not been as well studied as serine/threonine or tyrosine phosphorylation. We show that phosphohistidine and phosphohistidine-containing phosphopeptides derived from proteolytic digestion of phosphohistone H4 are detectable by ESI-MS. We also demonstrate reverse-phase HPLC separation of these phosphopeptides and their detection by MALDI-TOF-MS.     

Mass spectrometric identification of a novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I

Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) consists of at least 46 subunits. Phosphorylation of the 42-kDa subunit NDUFA10 was recently reported using a novel phosphoprotein stain [Schulenberg et al. (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J. Biol. Chem. 278, 27251]. Two smaller Complex I phosphoproteins, ESSS and MWFE, and their sites of modification, have since been determined [Chen et al. (2004) The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. 279, 26036]. Here we identify the site of phosphorylation in NDUFA10 from bovine heart mitochondria by tandem mass spectrometry. A single phosphopeptide spanning residues 47-60 was identified and confirmed by synthesis to be (47)LITVDGNICSGKpSK(60), establishing serine-59 as the site of phosphorylation.