Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS.

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Topical heparin as an effective and safe treatment for patients with capecitabine-induced hand-foot syndrome: results of a phase IIA trial supported by proteomic profiling of skin biopsies.

Background: Hand-foot syndrome (HFS) is a common adverse reaction associated with capecitabine chemotherapy that significantly affects the quality of life of patients. This study evaluates the safety and effectiveness of a topical heparin (TH) treatment on the clinical manifestations and anatomopathological alterations of capecitabine-induced HFS. In addition, we performed proteome profiling of skin biopsies obtained from patients with HFS at baseline and after heparin treatment. Methods: Patients with grade ⩽ 2 HFS associated with capecitabine were included in this study. The primary end point was the effectiveness of TH in reducing HFS of any grade. Clinical improvement was evaluated by clinicians, and an improvement was perceived by patients who performed a weekly visual analog scale questionnaire. Secondary end points included a comparative histological analysis and protein expression in skin biopsies at baseline and after 3 weeks of HT treatment. Proteomic profiling was carried out using quantitative isobaric labelling and subsequently validated by a T-array. Results: Twenty-one patients were included in the study. The median TH treatment time was 7.6 weeks (range = 3.6-41.6 weeks), and the median response time was 3.01 weeks (95% CI = 2.15-3.97). At the end of treatment, 19 of 21 patients (90.48%) responded to treatment with a decrease in one or more grades of HFS. None of the patients experienced adverse effects related to TH usage, nor did they suspend chemotherapy treatment. The main findings observed in skin biopsies after treatment were a decrease in hyperkeratosis and lymphocytic infiltrates. The proteomic analysis showed altered expression of 34 proteins that were mainly related to wound healing, cell growth, and the immune response. Conclusion: Based on our results, topical heparin is an effective and safe treatment for clinical manifestations of HFS, probably due to the restauration of skin homeostasis after heparin treatment, as supported by our proteomics-derived data. Trial registration: EudraCT 2009-018171-13.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation.

Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

On the Statistical Significance of Compressed Ratios in Isobaric Labeling: A Cross-Platform Comparison.

Isobaric labeling is gaining popularity in proteomics due to its multiplexing capacity. However, copeptide fragmentation introduces a bias that undermines its accuracy. Several strategies have been shown to partially and, in some cases, completely solve this issue. However, it is still not clear how ratio compression affects the ability to identify a protein's change of abundance as statistically significant. Here, by using the "two proteomes" approach (E. coli lysates with fixed 2.5 ratios in the presence or absence of human lysates acting as the background interference) and manipulating isolation width values, we were able to model isobaric data with different levels of accuracy and precision in three types of mass spectrometers: LTQ Orbitrap Velos, Impact, and Q Exactive. We determined the influence of these variables on the statistical significance of the distorted ratios and compared them to the ratios measured without impurities. Our results confirm previous findings1-4 regarding the importance of optimizing acquisition parameters in each instrument in order to minimize interference without compromising precision and identification. We also show that, under these experimental conditions, the inclusion of a second replicate increases statistical sensitivity 2-3-fold and counterbalances to a large extent the issue of ratio compression.

USP7 is a SUMO deubiquitinase essential for DNA replication.

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.

Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage.

Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC.

CUL4A contributes to the biology of basal-like breast tumors through modulation of cell growth and antitumor immune response.

The CUL4A E3 ubiquitin ligase is involved in the regulation of many cellular processes and its amplification and/or overexpression has been observed in breast cancer. The 13q34 amplification, which is associated with the basal-like breast cancer subtype, has been proposed as one of the mechanism behind CUL4A up-regulation. However, the specific contribution of CUL4A to the biology of basal-like breast tumors has not yet been elucidated. In this work, by using cellular models of basal phenotype, we show the inhibitory effect of CUL4A silencing in the proliferation and growth of breast cancer cells both, in vitro and in vivo. We also demonstrate the transforming capacity of CUL4A exogenous overexpression in the 184B5 human mammary epithelial cells in vitro. Our results suggest a synergistic effect between CUL4A high levels and the activation of the RAS pathway in the tumorigenesis of basal-like breast cancer tumors. In addition, by using a proteomics approach we have defined novel candidate proteins and pathways that might mediate the oncogenic effect of CUL4A. In particular, we report a putative role of CUL4A in bypassing the immune system in breast cancer through the down-regulation of several molecules involved in the immune surveillance. These findings provide insight into the oncogenic properties of CUL4A in basal-like breast cancer and highlight the therapeutic opportunities to target CUL4A.

S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3.

Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin. S100A9 is shown here to function as a chromatin component modulating C3 expression in mouse and human cells by binding to a region upstream of the C3 start site. When S100A9 was genetically deleted in mouse models of skin inflammation, the psoriasis-like skin disease and inflammation were strongly attenuated, with a mild immune infiltrate and decreased amounts of C3. In addition, inhibition of C3 in the mouse model strongly reduced the inflammatory skin disease. Thus, S100A8-S100A9 can regulate C3 at the nuclear level and present potential new therapeutic targets for psoriasis.

A proteomic characterization of factors enriched at nascent DNA molecules.

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.

Analysis of protein expression in developmental toxicity induced by MeHg in zebrafish.

Mercury toxicity and its implications in development are a major concern, due to the major threat to ecosystems and human health that this compound represents. Although some of the effects of methylmercury (MeHg) exposure have been extensively studied, the molecular mechanisms of interaction between this compound and developing organisms are still not completely understood. To provide further insights into these mechanisms, we carried out a quantitative proteomic study (iTRAQ) using zebrafish larvae exposed to 5 µg L(-1) and 25 µg L(-1) MeHg as a model. In this study, a multidimensional approach combining isoelectric focusing (IEF) and strong cation exchange (SCX) followed by reversed phase liquid chromatography prior to MALDI TOF/TOF analysis was employed, which resulted in a substantial increase in proteome coverage. Among the proteins identified, 71 were found de-regulated by more than 1.5-fold, and implicated in embryonic development, protein synthesis, calcium homeostasis and energy production. Furthermore, morphological and histological analysis of exposed larvae was carried out, reflecting changes such as smaller swim bladder, remaining yolk, bent body axis and accumulation of blood in the heart, among others.

Differential protein expression profiling by iTRAQ-two-dimensional LC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene.

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.