Catalin E. Doneanu

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents.

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co‐immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5‐signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule‐associated protein 2a and dynamin 1, as mGluR5‐interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.

Analysis of Oligosaccharides Derived from Heparin by Ion-Pair Reversed-Phase Chromatography/Mass Spectrometry

Current chromatographic and mass spectrometric techniques have limitations for analyzing heparin and heparin oligomers due to their high polarity, structural diversity, and sulfate lability. A rapid method for the analysis of heparin oligosaccharides was developed using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method utilizes an optimized buffer system containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), to achieve highly efficient separation together with enhanced mass response of heparin oligosaccharides. Analyses of a heparin oligosaccharide test mixture, dp6 through dp22, reveal that the chromatographic conditions enable baseline resolution of isomeric heparin oligosaccharides (dp6) and produce intact molecular ions with no sulfate losses during mass spectrometric analysis. In addition, the described conditions are amenable to the detection of heparin oligosaccharides in positive ion mode, yield stronger positive ion signals for corresponding oligosaccharides compared to the negative ion mode, and allow identification of structural isomers by an MS/MS approach. Because sensitive detection of oligosaccharides is also achieved with ultraviolet (UV) detection, the method utilizes a dual detection scheme (UV and MS in series) along with IPRP UPLC to simultaneously obtain quantification (UV) and characterization (MS) data for heparin oligosaccharides. The broad potential of this new method is further demonstrated for the analysis of a low-molecular-weight heparin (LMWH) preparation from porcine heparin. This approach will be of particular utility for profiling the molecular entities of heparin materials, as well as for structural variability comparison for samples from various sources.

Of mice and men: comparative proteomics of bronchoalveolar fluid.

We hypothesised that comparing the protein mixture in bronchoalveolar lavage fluid (BALF) between humans and mice may lead to mechanistic insights into common and divergent pathways that evolved in each species. BALF from four humans and six mice was pooled separately and underwent identical shotgun proteomic analysis. Functional and network analysis was applied to identify overlapping and distinct pathways enriched in the BALF. Follow-up experiments using Western analysis in unpooled BALF samples were performed. We identified 91 unique proteins in human and 117 unique proteins in mouse BALF samples. Functional analysis of the proteins revealed conservation of several key processes between the species, including defence response. Oxidative stress response, however, was selectively enriched only in mouse BALF. Differences in the expression of peroxiredoxin-1, a key member of the defence pathway against oxidative injury, were confirmed between normal human and mouse BALF and in models of lung injury. A computational proteomics approach of mouse and human BALF confirms the conservation of immune and defence-mediated pathways while highlighting differences in response to oxidative stress. These observations suggest that the use of mice models to study human lung disorders should be undertaken with an appreciation of interspecies variability.

Identification of the interactions between cytochrome p450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis

The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b5 (b5) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an 18O-labeling method on the basis that cross-linked peptides incorporate twice as many 18O atoms as non-cross-linked peptides during proteolysis conducted in 18O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys428(CYP2E1)-Asp53(b5) and Lys434(CYP2E1)-Glu56(b5), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b5 interaction, and the stimulatory effect of b5, was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b5 complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.

Identification and type III‐dependent secretion of the Yersinia pestis insecticidal‐like proteins

Plague, or the Black Death, is a zoonotic disease that is spread from mammal to mammal by fleas. This mode of transmission demands that the causative agent of this disease, Yersinia pestis, is able to survive and multiply in both mammals and insects. In recent years the complete genome sequence of a number of Y. pestis strains have been determined. This sequence information indicates that Y. pestis contains a cluster of genes with homology to insecticidal toxin encoding genes of the insect pathogen Photorhabdus luminescens. Here we demonstrate that Y. pestis KIM strains produced the encoded proteins. Production of the locus‐encoded proteins was dependent on a gene (yitR) encoding a member of the LysR family of transcriptional activators. Evidence suggests the proteins are type III secretion substrates. N terminal amino acids (100 to 367) of each protein fused to an epitope tag were secreted by the virulence plasmid type III secretion type. A fusion protein comprised of the N‐terminus of YipB and the enzymatic active component of Bordetella pertussis adenylate cyclase (Cya) was translocated into both mammalian and insect cells. In conclusion, a new class of Y. pestis type III secreted and translocated proteins has been identified. We hypothesize that these proteins function to promote transmission of and infection by Y. pestis.

Age-Related Increase in 4-Hydroxynonenal Adduction to Rat Heart α-Ketoglutarate Dehydrogenase Does Not Cause Loss of Its Catalytic Activity

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (p< or =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (p< or =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (p< or =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the authors laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

Identification of secreted glycoproteins of human prostate and bladder stromal cells by comparative quantitative proteomics.

Functional development of the prostate is governed by stromal mesenchyme induction and epithelial response. Stromal/epithelial signaling can be mediated through direct cell–cell contact and diffusible factors and their cell surface receptors. These inducers are likely secreted or membrane‐associated extracellular proteins. Given the importance of intercellular communication, it is possible that diseases like cancer could arise from a loss of this communication. One approach to gain a molecular understanding of stromal cells is to identify, as a first step, secreted stromal signaling factors. We proposed to do this by comparative analysis between bladder and prostate.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin −198 Mutation in the γ-Globin Gene Promoter

Expression of the γ-globin gene is silenced in adult humans. However, certain point mutations in the γ-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T →C point mutation at position –198 of the Aγ-globin gene promoter results in 4–10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH –198 γ-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH –198 γ-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of γ-globin gene expression in the adult transcriptional environment is discussed.