A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved ...digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
LC-MS analysis of therapeutic antibodies and other biotherapeutics from in-life studies (e.g., serum/plasma) has evolved from simple peptide digestion to peptide mapping and intact mass monitoring. ...From more advanced analytical approaches, a deeper understanding as to the fate of the biotherapeutic in vivo is gained. Here, we examine the next generation of approaches to facilitate the most comprehensive understanding of large molecule drug fate in circulation. Three case studies are presented: (1) use of relative and absolute calibration curves for biotherapeutic quantitation from the same sample set; (2) top-down mass spectrometry applied to bioanalytical assays; (3) biotherapeutic protein complexes from serum analyzed by native protein MS. We anticipate that these approaches will be further adapted and applied by other research groups.
A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to ...test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).
Degree of labeling and label efficiency are key factors for optimal characterization of critical reagents that are used in ligand binding assays. Here, three case studies are shown demonstrating how ...liquid chromatography–mass spectrometry (LC-MS) was utilized to characterize critical reagents using three unique methodologies. Critical reagent batches were prepared for LC-MS analysis by use of: 20 mM dithiothreitol (DTT) (Case 1), rapid PNGaseF (Case 2), and a mobile phase diluent (Case 3). LC-MS was run at three different MS method conditions in each troubleshooting case specific for reduced IgG, intact IgG, and native LC-MS, respectively. Specified LC-MS methods based on sample type and configuration elucidated clear MS profiles, allowing for degree of labeling and label efficiencies to be calculated. Ultimately the LC-MS analyses were fine-tuned for critical reagent characterization, and practices for analyzing similar reagents in the future can be established.
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•Three LC-MS methods specified for characterization of unique critical reagents.•Reduced mAb LC-MS solved assay signal issue for capture reagent.•Multiple batches of a heavily glycosylated ruthenium labeled reagent resolved for reagent consistency.•Native MS method analyzed a labeled capture reagent with artificial mAb backbone.•DOL and label efficiency values from LC-MS assisted in critical reagent consistency in LBA's.
Recent advancements in immunocapture methods and mass spectrometer technology have enabled intact protein mass spectrometry to be applied for the characterization of antibodies and other large ...biotherapeutics from in-life studies. Protein molecules have not been traditionally studied by intact mass or screened for catabolites in the same manner as small molecules, but the landscape has changed. Researchers have presented methods that can be applied to the drug discovery and development stages, and others are exploring the possibilities of the new approaches. However, a wide variety of options for assay development exists without clear recommendation on best practice, and data processing workflows may have limitations depending on the vendor. In this perspective, we share experiences and recommendations for current and future application of mass spectrometry for biotherapeutic molecule monitoring from preclinical and clinical studies.
Constitutive production of inflammatory cytokines is a characteristic of many human malignant cell lines; however, the in vitro and in vivo interdependence of these cytokines, and their significance ...to the human cancer microenvironment, are both poorly understood. Here, we describe for the first time how three key cytokine/chemokine mediators of cancer-related inflammation, TNF, CXCL12, and interleukin 6, are involved in an autocrine cytokine network, the "TNF network," in human ovarian cancer. We show that this network has paracrine actions on angiogenesis, infiltration of myeloid cells, and NOTCH signaling in both murine xenografts and human ovarian tumor biopsies. Neutralizing antibodies or siRNA to individual members of this TNF network reduced angiogenesis, myeloid cell infiltration, and experimental peritoneal ovarian tumor growth. The dependency of network genes on TNF was shown by their downregulation in tumor cells from patients with advanced ovarian cancer following the infusion of anti-TNF antibodies. Together, the findings define a network of inflammatory cytokine interactions that are crucial to tumor growth and validate this network as a key therapeutic target in ovarian cancer.
Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-alpha is an important component of this ...interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-alpha was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-alpha-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25- cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-alpha signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-kappaB system, TGF-beta1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-alpha in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.
Time-of-flight MS systems for biopharmaceutical and protein characterization applications may play an even more pivotal role in the future as biotherapeutics increase in drug pipelines and as ...top-down MS approaches increase in use. Here, a recently developed TOF MS system is examined for monoclonal antibody (mAb) characterization from serum samples. After immunocapture, purified drug material spiked into monkey serum or dosed for an in-life study is analyzed by top-down MS. While characterization aspects are a distinct advantage of the MS platform, MS system and software capabilities are also shown regarding intact protein quantitation. Such applications are demonstrated to help enable comprehensive protein molecule quantitation and characterization by use of TOF MS instrumentation.
Focusing on the semi-arid and highly disturbed landscape of San Clemente Island (SCI), California, we test the effectiveness of incorporating a hierarchical object-based image analysis (OBIA) ...approach with high-spatial resolution imagery and canopy height surfaces derived from light detection and ranging (lidar) data for mapping vegetation communities. The hierarchical approach entailed segmentation and classification of fine-scale patches of vegetation growth forms and bare ground, with shrub species identified, and a coarser-scale segmentation and classification to generate vegetation community maps. Such maps were generated for two areas of interest on SCI, with and without vegetation canopy height data as input, primarily to determine the effectiveness of such structural data on mapping accuracy. Overall accuracy is highest for the vegetation community map derived by integrating airborne visible and near-infrared imagery having very high spatial resolution with the lidar-derived canopy height data. The results demonstrate the utility of the hierarchical OBIA approach for mapping vegetation with very high spatial resolution imagery, and emphasizes the advantage of both multi-scale analysis and digital surface data for accurately mapping vegetation communities within highly disturbed landscapes.
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