Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.
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PMID:Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator. 852 Jun 84

High-performance anion-exchange chromatography (HPAEC) with pulsed-amperometric detection was used to monitor the consistency of the oligosaccharides released from several partially purified preparations of a tissue-type plasminogen activator mutant (TNK-tPA). Differences in the oligosaccharide map were observed, primarily in the neutral carbohydrate region of the separation. Subsequent investigations using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) identified the neutral oligosaccharides to be primarily asialo-diantennary complex-type glycans with 2, 1, or 0 galactose residues. Additional asialo-triantennary and asialo-tetrantennary structures were also observed with varying amounts of galactose. Hydrolysis of the chromogenic substrate 4-methyl umbelliferyl-beta-galactoside confirmed that host-cell lysosomal beta-galactosidase is present at the first step in the purification process and is the cause of the observed glycan degradation. Subsequent steps in the purification process quantitatively remove this enzyme. Comparison of HPAEC and MALDI/TOF/MS analysis of time-course samples revealed quite similar rates of degradation and demonstrates the quantitative utility of these methods. HPAEC did not reveal significant changes in the sialylated structures as evidenced by nearly identical profiles for the sialic acid-containing structures.
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PMID:The use of high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor and identify oligosaccharide degradation. 866 Jun 30

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.
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PMID:Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 906 96

The N-linked glycans assembled in Pichia pastoris on the recombinant kringle 2 domain of human tissue-type plasminogen activator (r-[K2tPA]) are composed of approx. 80% neutral and 20% charged species. After peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase-catalysed liberation of the oligosaccharides from the purified glycopeptide, the glycan mixture was resolved by HPLC on amino-silica-based resin. Oligosaccharide mapping of the resulting mixture by HPLC, gel filtration and time-of-flight matrix-assisted laser-desorption-ionization-with-delayed-extraction mass spectrometry (TOF-MALDI DE-MS) revealed that > 90% of the charged species consisted of a series of oligosaccharides possessing molecular masses that were consistent with a range of saccharides comprising phospho-Man10GlcNAc2-phospho-Man14GlcNAc2, with phospho-Man11GlcNAc2 representing the major species. The remaining material in the charged fraction contained identifiable phosphorylated glycans that were one or two mannose units shorter, and one to four mannose units longer, than those present in the above range of oligosaccharides. Treatment of the native charged glycan pool with alkaline phosphatase did not result in molecular-size alterations, showing that phosphomonoesters are not present. Mild acid hydrolysis of the glycans led to a decrease in the size of all charged glycans by one mannose residue, providing phospho-Man9GlcNAc2-phospho-Man13GlcNAc2. Following this procedure, treatment with alkaline phosphatase resulted in size decreases that were equivalent to the loss of one phosphate group from each glycan. This demonstrates that all charged glycans isolated contained phosphate in phosphodiester bonds to two mannose units. The present study shows that P. pastoris cells possess the capability of assembling phosphorylated glycans having the phosphate moiety present in phosphodiester linkages with two mannose units. These saccharides, like the neutral oligosaccharides, contain considerably smaller amounts of mannose than glycans present in other strains of yeast.
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PMID:Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator. 935 3

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.
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PMID:A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. 959 42

Process development for biopharmaceuticals is dictated by product quality, drug safety and economy of the manufacturing process. Not surprisingly, these factors also play a key role in the evaluation of mammalian cell expression systems to be used in the production of pharmacologically active glycoproteins. To date, the most prominent candidates for efficient expression of glycoproteins are mammalian cell lines such as mouse fibroblast cells (C 127-BPV), Chinese hamster ovary cells (CHO-DHFR, CHO-NEOSPLA, CHO-GS), mouse myeloma cells (NSO-GS) as well as transgenic animals carrying c-DNA or genomic DNA which codes for the protein of interest. The expression titer in the case of glycoproteins is mainly determined by the promoter construct, the site of integration into the chromosome, the copy number and the type of protein in question. Based on expression titer, CHO-NEOSPLA and NSO-GS expression systems are most effective in the production of monoclonal antibodies and, to a lesser extent, of recombinant DNA derived proteins. However, based on overall product yield, expression of recombinant DNA derived proteins in transgenic animals is by far the most promising system. Therefore, for proteins required in large quantities, transgenic expression systems offer an attractive choice. However, cost of goods for products for which the dosage or the overall annual quantities are low, is dominated by downstream processing, filling, lyophilization and packaging and not by the fermentation process. Such proteins are preferentially produced by classical mammalian cell culture systems. Concerns which have to be addressed with respect to drug safety in the transgenic animal approach are the size of the herd, genetic stability from animal to animal, variation in productivity and in impurity profiles during lactation periods, microbial, viral, mycoplasma and prion contaminants, the dependence on health status and the life span of the animal. In a number of cases glycosylation of the protein is relevant for the prevention of immunogenicity of the protein, the pharmacological activity, the pharmacokinetic profile, solubility and stability against proteolysis. The glycosylation pattern, depending on protein structure, is influenced by the enzymatic system of the host cell as well as by fermentation conditions. Therefore, selection of host cells and culture conditions must take into account the requirement for a specific and stable glycosylation pattern. For the assessment of glycovariants, a number of protein analytical methods such as peptide mapping, isoelectric focusing, oligosaccharide mapping, MALDI-TOF (matrix assisted laser desorption mass spectrometry-time of flight), capillary electrophoresis and specific potency assays are available. In our experiments, glycosylation of proteins expressed in CHO cells was demonstrated to be very stable. Only extreme process times, cultivation methods and ammonium ion concentrations had an influence on the glycosylation profile. Among the three products investigated--tissue plasminogen activator (t-PA), interferon omega and soluble intercellular adhesion molecule (s-ICAM)--t-PA expressed the most stable glycosylation pattern. Only at extreme ammonium concentrations an increase of mannose-5 structures was observed, whereas biantennary complex structures were reduced. On the other hand, interferon omega and s-ICAM showed greater susceptibility to increased ammonium concentrations and to adherent cultivation. Such conditions induced quantitative changes to the glycosylation pattern favoring the appearance of higher branched structures. Short cultivation times resulted in more heterogenous oligosaccharide structures. Since the glycosylation of the three proteins is different in the same host cell, the amino acid sequence of the protein apparently influences the glycosylation pattern and its sensitivity to culture conditions. In NSO-mouse myeloma cells, production of s-ICAM is two times as high as in CHO cells
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PMID:Appropriate mammalian expression systems for biopharmaceuticals. 974 18

PLA(2)(EC3.1.1.4) of Agkistrodon blomhoffii Ussurensis of Changbai Mountain has been purified to homogeneity via four steps consisting of DEAE-Sephadex A-50, and three times Sephadex G-75 column chromatography in series. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization, time of flight mass spectrometry, the PLA(2) was found to have a molecular weight of (14.008+/-0.007) kD. The optimal pH of the PLA(2) is in the range from 8.0 to 9.0, the optimal temperature is 45 degrees. Polymer is found in solution by MALDI/TOF/MS.
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PMID:Purification and Characterization of A Phospholipase A(2) from Agkistrodon blomhoffii Ussurensis of Changbai Mountains. 1214 27

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.
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PMID:Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2). 1223 64

A polymeric micelle drug delivery system was developed to enhance the solubility of poorly-water soluble drug, biphenyl dimethyl dicarboxylate, DDB. The block copolymers consisting of poly(D,L-lactide) (PLA) as the hydrophobic segment and methoxy poly(ethylene glycol) (mPEG) as the hydrophilic segment were synthesized and characterized by NMR, DSC and MALDI-TOF mass spectroscopy. The size of the polymeric micelles measured by dynamic light scattering showed a narrow monodisperse size distribution with the average diameter less than 50 nm. The MW of mPEG-PLA, 3000 (MW of mPEG, 2 K; MW of PLA, 1 K), and the presence of hydrophilic and hydrophobic segments on the polymeric micelles were confirmed by MALDI-TOF mass spectroscopy and NMR, respectively. Polymeric micelle solutions of DDB were prepared by three different methods, i.e. the matrix method, emulsion method and dialy-sis method. In the matrix method, DDB solubility was reached to 13.29 mg/mL. The mPEG-PLA 2K-1 K micelle system was compared with the poloxamer 407 micelle system for their critical micelle concentration, micelle size, solubilizing capacity, stability in dilution and physical state. DDB loaded-polymeric micelles prepared by the matrix method showed a significantly increased aqueous solubility (>5000 fold over intrinsic solubility) and were found to be superior to the poloxamer 407 micelles as a drug carrier.
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PMID:A polymeric micellar carrier for the solubilization of biphenyl dimethyl dicarboxylate. 1264 97

The purpose of this study was to prepare poly(ethylene glycol) (PEG)ylated octreotide and investigate the stability against acylation by polyester polymers such as poly(lactic acid) and poly(lactic-co-glycolic acid). Octreotide was modified by reaction with monomethoxy PEG-propionaldehyde (molecular weight 5,000) in the presence of sodium cyanoborohydride. The mono-PEGylated fraction was isolated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Circular dichroism demonstrated no significant secondary structural differences between mono-PEGylated octreotide (mono-PEG-octreotide) and intact octreotide. As a test system for the stability study against acylation reaction, lactic acid (LA) solutions with various concentrations and pH values were prepared with water dilution and subsequent accelerated equilibration at 90 degrees C for 24 hours. Native octreotide was found to be acylated in all the diluted LA solutions with different concentrations (42.5%, 21.3%, and 8.5%, wt/wt) and pH values (2.25, 1.47, and 1.85, respectively). The remaining amounts of intact octreotide continuously decreased to 50% through 30 days of incubation at 37 degrees C. MALDI-TOF MS identified the octreotide to be acylated by LA units. However, acylation reaction of mono-PEG-octreotide in LA solutions was negligible, and the remaining amounts of intact one through 30 days of incubation in LA solutions were also comparable to the initial concentration. These data suggest that mono-PEG-octreotide may prevent the acylation reaction in degrading PLA microspheres and possibly serve as a new source for somatostatin microsphere formulation.
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PMID:Preparation and stability of poly(ethylene glycol) (PEG)ylated octreotide for application to microsphere delivery. 1519 67


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