Defining 'full-length' recombinant factor VIII: a comparative structural analysis.

Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.

Diagnostic accuracy of focused appendiceal CT in clinically equivocal cases of acute appendicitis.

PURPOSE: To determine the diagnostic accuracy of modified focused appendiceal computed tomography (CT) to exclude or confirm appendicitis in patients who presented with equivocal symptoms and signs of appendicitis. MATERIALS AND METHODS: One hundred patients (age range, 14-81 years; mean age, 30.6 years) with equivocal symptoms and signs of acute appendicitis were included in this prospective study. Patients were given 30 mL of diatrizoate meglumine and diatrizoate sodium and 60 mL of sorbitol mixed in 1 L of water orally over 1 hour. CT was performed 1.5 hours after the commencement of oral contrast material administration. The criteria used for the diagnosis of appendicitis were (a) appendix greater than 6 mm in maximum diameter, (b) no contrast material in the appendiceal lumen, and (c) inflammatory changes in the periappendiceal fat. CT results were compared with histopathologic findings at appendectomy. Patients with negative CT findings were followed up by telephone or clinically. RESULTS: Of 100 cases, 30 were positive at CT and 70 were negative. There were 28 true-positive cases; two false-positive cases, one cecal diverticulitis and one pelvic peritonitis with periappendicitis; and two false-negative cases, one perforated appendix and one mucosal and submucosal inflammation of the appendix but no transmural inflammation. Sensitivity was 93%, specificity was 97%, and accuracy was 96%. CONCLUSION: Focused appendiceal CT in which oral contrast material is used alone yields high levels of accuracy in clinically equivocal cases of acute appendicitis.

Identification of human serum interferants in the recombinant P-selectin glycoprotein ligand-1 clinical ELISA using MALDI MS and RP-HPLC.

A colorimetric enzyme-linked immunosorbent assay (ELISA) was developed to detect circulating levels of rPSGL to permit pharmacokinetic analysis of clinical samples. The ELISA is an asymmetric sandwich utilizing a monoclonal antibody pair. Initial validation studies indicated that 57% of normal individuals scored above the limit of detection of the assay. Specificity experiments indicated that the signal was not due to circulating endogenous P-selectin glycoprotein ligand-1 (PSGL-1). Using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and sampling within the individual microplate wells, the interferant was detected in the vicinity of 6.6 kDa in lipemic and normal human sera, but not delipidized sera. These results were consistent with the ELISA data where 97.5% of known lipemic, 57% of normal, and 0% of delipidized sera scored above detectable limits in the ELISA. Preparative isolations of the 6.6 kDa species were performed using reversed-phase high performance liquid chromatography (RP-HPLC) with UV and MS detection. Edman N-terminal sequencing identified the 6.6 kDa unknown as Apolipoprotein C-I. Additional apolipoproteins were found by MALDI and RP-HPLC. Digestion of sera with liposome lipase and extraction of sera with anti-apolipoprotein C-I, C-II, and C-III antibody beads significantly reduced the ELISA interference. These experiments combined with the MALDI detection of phosphatidylcholine-type lipids from NHS eluate suggested that lipoprotein particles or remnants were causing the interference. A method combining Triton-X 100 with sonication was developed to overcome this interference without altering rPSGL recovery in the ELISA.

Isomeric differentiation of asparagine-linked oligosaccharides by matrix-assisted laser desorption-ionization postsource decay time-of-flight mass spectrometry.

Matrix-assisted laser desorption-ionization (MALDI)-postsource decay (PSD) was used to differentiate glycoprotein-released N-linked oligosaccharide isomers directly from aliquots of glycosidase digests and peak fractions collected from high-pH anion exchange chromatography (HPAEC) with minimal sample handling and material. With the implementation of instrumental tuning and acquisition controls, MALDI-PSD of NMR-characterized high-mannose, hybrid, and complex standards resulted in spectra with reproducible fragment ion peak intensity ratios which correlated well to the respective oligosaccharide branching patterns. A "knowledge-based" strategy was utilized to characterize unknown isomeric N-glycan structures in which specific fragment ion types and their distributions in the unknown PSD spectrum were compared to those in PSD spectra of standards possessing similar structural features. This PSD knowledge-based isomeric differentiation strategy was applied to distinguishing recombinant glycoprotein-derived Man7 D1 versus D2/D3 isomers directly from a PNGaseF digest aliquot of high-mannose N-glycans based on branching differences. A precursor ion selection device was employed to isolate the component of interest from the mass profile without additional chromatographic isolation steps. MALDI-MS signal-to-background was maximized for direct PSD with on-the-probe sample clean-up methods. The asialo complex N-glycan PSD knowledge base was used to differentiate HPAEC peak fractions containing the tri- and tri'-antennary branching isomers and two tetraantennary isomers with antennal versus core fucose locations. Although the particular asialo complex N-glycan isomers here were well separated by HPAEC, MALDI-MS alerted us to their presence using m/z-derived monosaccharide compositions and PSD fragmentation allowed us to differentiate these structures using the HPAEC elution positions as guides.

On-the-probe sample cleanup strategies for glycoprotein-released carbohydrates prior to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

On-the-probe sample cleanup strategies were developed for matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to improve the mass spectral characteristics of glycoprotein-released carbohydrate samples, including those fractionated by high pH anion exchange (HPAE) chromatography or treated with glycosidases. Small in situ amounts of chromatographic media are codeposited with matrix onto a probe containing a carbohydrate sample to minimize interferences from cations, anions, and/or detergents introduced from the sample and/or matrix. On-the-probe sample cleanup is fast (a few minutes) and operates best on picomole quantities of analyte in sample volumes less than 5 microliter containing nanomole quantities or less of impurities. This in situ cleanup dramatically increases the mass spectral signal-to-background, improves mass accuracies, better equalizes the sensitivities for diverse carbohydrate structures, and has the potential to remove contaminants that bypassed previous purification schemes. Direct MALDI mass profiling of digest aliquots containing low picomole amounts of carbohydrate structures either enzymatically released from a glycoprotein or sequentially degraded with multiple glycosidases was performed using only microscale digest conditions with reduced buffer amounts and on-the-probe sample cleanup to minimize the digest impurities. Membrane microdialysis was compared to on-the-probe sample cleanup and found to more completely remove the nano- to micromole amounts of anions (and cations) in HPAE fractions in one step as opposed to multiple on-the-probe steps.

Injection of reagent ions into the selvedge region in fast-atom bombardment mass spectrometry.

A divided probe that incorporates a potassium aluminosilicate glass target and an analyte/glycerol matrix target, spatially separated, was used to inject potassium ions (K(+)) into the high-pressure "selvedge" region formed above the analyte/glycerol matrix target during fast-atom bombardment (FAB); [M+K](+) adduct ions that represent the types of gas-phase neutral molecules present in the selvedge region are observed. Computer modeling assisted in designing the divided target and an additional ion optical element for the FAB ion source to optimize interactions between K(+) ions and the desorbed neutral molecules. The capability of injecting K(+) ions into the FAB experiment has utility in both mechanistic studies and analyses. Experimental results here are consistent with a model for the desorption/ionization processes in FAB in which some types of neutral analyte molecules are desorbed intact and are subsequently protonated by glycerol chemical ionization. Unstable protonated molecules undergo unimolecular decomposition to yield observed fragment ions. The use of K(+) cationization of analytes for molecular weight confirmation is demonstrated, as well as its utility in FAB experiments in which mixtures are encountered.