Human serum albumin~HSA! interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and0or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.

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Hope rests on the envelope proteins of human immunodeficiency virus (HIV) as protective vaccines and thus their antibody binding sites are of prime interest. 2G12 and other human antibodies bind to a cluster of oligomannose N-glycans. Owing to the extreme number and density of N-glycosylation sites gp160 and its recombinant form gp140 represent challenging tasks for site-specific glycosylation analysis. We have conducted a glycosylation analysis of CN54gp140 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using an ion trap as well as a Q-TOF instrument and standard software for glycopeptide identification. First, a deglycosylated sample of the protease digest served to locate the elution positions of peptides covering all of the 27 potential N-glycosylation sites. Then, the assignments of the similarly eluting glycopeptides were verified by collision-induced decay MS/MS experiments with elevated fragmentation energy. The acquisition of site-specific glycan profiles was facilitated by the use of buffered eluent, which rounds up all glycoforms of a peptide into one peak. Calculation of the molecular mass drawn on the weighted averages of the glycans at each site led to the actual mass of gp140 of approximately 120 kDa.

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

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Recombinant human follicle stimulating hormone is an important drug in reproductive medicine. Thorough analysis of the heterodimeric heavily glycosylated protein is a prerequisite for the evaluation of production batches as well as for the determination of "essential similarity" of new biosimilars. The concerted application of different liquid chromatography-mass spectrometry methods enabled the complete depiction of the primary structure of this pituitary hormone. Sequence coverage of 100% for the α- as well as the β-chain was achieved with tryptic peptides. Most of these peptides could be verified by tandem mass spectrometry. Site-specific analysis of all four glycosylation sites was, however, not possible with tryptic but with chymotryptic peptides. Quantification of the glycoforms of each glycopeptide was accomplished with the software MassMap®. Both protein subunits gave interpretable mass spectra upon S-alkylation and separation on a C5 reversed-phase column. Glycan isomer patterns were depicted by separation on porous graphitic carbon, using mass spectrometric detection for the evaluation of the glycopeptide liquid chromatography-electrospray ionization data. The currently marketed product Gonal-f™ and a potential biosimilar were compared with the help of these procedures.

Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.