Next generation proteomics in precision oncology: 1000s of proteome and phosphoproteome profiles of tumors and matching healthy tissues as meaningful layer in multi-omics database.

e15672 Background: The rise of precision oncology therapeutics requires deep understanding of all molecular mechanisms involved in cancer biology. IndivuType offers the world’s first multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics (WGS), transcriptomics, proteomics, and clinical outcome information. Indivumed is committed to the quality of the IndivuType ecosystem starting with stringent SOP-driven sample collection combined with thorough validation of clinical information and data integrity. The availability of multi-omics data from the same tumor can provide a comprehensive molecular picture of cancer for a given patient. Protein expression and activation are directly related to cellular function and hence provide actionable information about druggable targets. Until recently, the proteomics technology could not match the scale of next-gen sequencing and consequently precision medicine has almost exclusively been based on gene level data. Here we present the first large-scale data set for protein expression and phosphorylation. Enabled by the data independent acquisition (DIA) workflow, a mass spectrometric method provided by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome (WP) and 20,000 phospho-peptides in the phospho-proteome (PP) workflow were profiled. Methods: Sample processing from 5 mg of tissue per sample was performed using liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. DIA LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (WP) and 60 min (PP) gradients. Results: Several thousands of high-quality patient samples of various cancer types have been analyzed to date. The resulting proteome and phospho-proteome data has been integrated into the IndivuType database, thereby providing a solid foundation to advance our understanding of cancer. Conclusions: With the ongoing addition of more samples and associated deep and rich data, the platform could unravel key molecular events and is expected to transform knowledge into actionable treatments and personalized therapies.

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Proteome and phospho-proteome profiling for deeper phenotype characterization of colorectal cancer heterogeneity.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15536 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15536-e15536

Author(s):

Jakob Vowinckel ◽

Thomas Corwin ◽

Jonathan Woodsmith ◽

Tobias Treiber ◽

Roland Bruderer ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Biology ◽

Molecular Mechanisms ◽

Deep Understanding ◽

Peptide Fragmentation ◽

Spectrometric Method ◽

Molecular Heterogeneity ◽

Precision Oncology ◽

Mass Spectrometric Method

e15536 Background: The rise of precision oncology therapeutics requires deep understanding of the molecular mechanisms implicated in cancer biology. Colorectal cancer (CRC) is one of the first solid tumors to be molecularly characterized by defined genes and pathways. Advances in tumor profiling have revealed a profound molecular heterogeneity in CRC leading to the definition of several consensus molecular subtypes (CMS). However, this molecular heterogeneity is still largely defined on the genomic and transcriptomics level. To complement the understanding of genetically defined molecular subgroups, we performed large-scale deep proteomic and phospho-proteomic profiling of CRC patient biopsies and adjacent healthy control tissue, which has enabled to explore the phenotype and obtain more functional insights in cancer biology. Methods: Sample processing from 5-10 mg of tissue per sample was performed using a liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. Data-Independent Acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (whole proteome) and 60 min (phospho-proteome) gradients. Results: Indivumed has built IndivuType, the world’s first multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics, transcriptomics, proteomics, and clinical outcome information. Enabled by the DIA technology, a mass spectrometric method developed by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome and 20,000 phospho-peptides in the phospho-proteome workflow were profiled across more than 900 resected tissue samples of various CMS of CRC. The resulting proteome and phospho-proteome data were integrated into the IndivuType database and cross-analyzed with genomic and transcriptomic markers. Through this combined analysis, novel insights in clinically relevant signaling pathways in CRC subtypes were revealed. Conclusions: The deep phenotypic profiling of cancer samples, using next generation proteomics and phospho-proteomics, has enabled us to go beyond the genomic level in the characterization of tumor molecular heterogeneity. This multi-omics approach provides a solid foundation to advance the understanding of cancer biology, unravel key molecular events, and support the identification of novel therapeutic targets for precision medicine in CRC.

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Systematic detection of functional proteoform groups from bottom-up proteomic datasets

10.1101/2020.12.22.423928 ◽

2020 ◽

Author(s):

Isabell Bludau ◽

Max Frank ◽

Christian Dörig ◽

Yujia Cai ◽

Moritz Heusel ◽

...

Keyword(s):

Alternative Splicing ◽

Molecular Mechanisms ◽

Data Matrix ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Bottom Up ◽

Tissue Samples ◽

Protein Coding ◽

Systematic Assessment ◽

A Genome

AbstractThe cellular proteome, the ensemble of proteins derived from a genome, catalyzes and controls thousands of biochemical functions that are the basis of living cells. Whereas the protein coding regions of the genome of the human and many other species are well known, the complexity and composition of proteomes largely remains to be explored. This task is challenging because mechanisms including alternative splicing and post-translational modifications generally give rise to multiple distinct, but related proteins – proteoforms – per coding gene that expand the functional capacity of a cell.Bottom-up proteomics is a mass spectrometric method that infers the identity and quantity of proteins from the measurement of peptides derived from these proteins by proteolytic digestion. Whereas bottom-up proteomics has become the method of choice for the detection of translation products from essentially any gene, the inherent missing link between measured peptides and their parental proteins has so far precluded the systematic assessment of proteoforms and thus limited the resolution of proteome maps. Here we present a novel, data-driven strategy to assign peptides to unique functional proteoform groups based on peptide correlation patterns across large bottom-up proteomic datasets. Our strategy does not fully characterize specific proteoforms, as is achievable in top-down approaches. Rather, it clusters peptides into functional proteoform groups that are directly linked to the biological context of the study. This allows the detection of tens to hundreds of proteoform groups in an untargeted fashion from bottom-up proteomics experiments.We applied the strategy to two types of bottom-up proteomic datasets. The first is a protein complex co-fractionation dataset where native complexes across two different cell cycle stages were resolved and analyzed. Here, our approach enabled the systematic detection and evaluation of assembly specific proteoforms at an unprecedented scale. The second is a protein abundance vs. sample data matrix typical for bottom-up cohort studies consisting of tissue samples from the mouse BXD genetic reference panel. In either data type the method detected state-specific proteoform groups that could be linked to distinct molecular mechanisms including proteolytic cleavage, alternative splicing and phosphorylation. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from bottom-up proteomic datasets.

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Celecoxib repurposing in cancer therapy: molecular mechanisms and nanomedicine-based delivery technologies

Nanomedicine ◽

10.2217/nnm-2021-0086 ◽

2021 ◽

Author(s):

Asmaa F Khafaga ◽

Rehab N Shamma ◽

Ahmed Abdeen ◽

Abdelmonem M Barakat ◽

Ahmed E Noreldin ◽

...

Keyword(s):

Cancer Therapy ◽

Anticancer Agents ◽

Cancer Biology ◽

Molecular Mechanisms ◽

Deep Understanding ◽

Drug Repurposing ◽

Special Focus ◽

Drug Classes ◽

Cox 2 ◽

Cox 2 Inhibitors

While cancer remains a significant global health problem, advances in cancer biology, deep understanding of its underlaying mechanism and identification of specific molecular targets allowed the development of new therapeutic options. Drug repurposing poses several advantages as reduced cost and better safety compared with new compounds development. COX-2 inhibitors are one of the most promising drug classes for repurposing in cancer therapy. In this review, we provide an overview of the detailed mechanism and rationale of COX-2 inhibitors as anticancer agents and we highlight the most promising research efforts on nanotechnological approaches to enhance COX-2 inhibitors delivery with special focus on celecoxib as the most widely studied agent for chemoprevention or combined with chemotherapeutic and herbal drugs for combating various cancers.

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Tandem Mass Spectrometric Evaluation of Core Structures of Aromatic Compounds after Catalytic Deoxygenation

10.26434/chemrxiv.5410807.v1 ◽

2017 ◽

Author(s):

Xueming Dong

Keyword(s):

Aromatic Compounds ◽

Supported Catalyst ◽

Mass Spectrometric ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Aromatic Rings ◽

Tandem Mass Spectrometric ◽

Catalytic Deoxygenation ◽

Oxygen Atoms

Catalytic deoxygenation of coal enhances the stability and combustion performance of coal-derived liquids. However, determination of the selectivity of removal of oxygen atoms incorporated in or residing outside of aromatic rings is challenging. This limits the ability to evaluate the success of catalytic deoxygenation processes. A mass spectrometric method, in-source collision-activated dissociation (ISCAD), combined with high resolution product ion detection, is demonstrated to allow the determination of whether the oxygen atoms in aromatic compounds reside outside of aromatic rings or are part of the aromatic system, because alkyl chains can be removed from aromatic cores via ISCAD. Application of this method for the analysis of a subbituminous coal treated using a supported catalyst revealed that the catalytic treatment reduced the number of oxygen-containing heteroaromatic rings but not the number of oxygen atoms residing outside the aromatic rings.<br>

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Tandem Mass Spectrometric Evaluation of Core Structures of Aromatic Compounds after Catalytic Deoxygenation

10.26434/chemrxiv.5410807 ◽

2017 ◽

Author(s):

Xueming Dong

Keyword(s):

Aromatic Compounds ◽

Supported Catalyst ◽

Mass Spectrometric ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Aromatic Rings ◽

Tandem Mass Spectrometric ◽

Catalytic Deoxygenation ◽

Oxygen Atoms

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Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180110141827 ◽

2018 ◽

Vol 18 (7) ◽

pp. 1025-1031

Author(s):

Cheng Luo ◽

Di Wu ◽

Meiling Chen ◽

Wenhua Miao ◽

Changfeng Xue ◽

...

Keyword(s):

Cell Proliferation ◽

Confocal Microscopy ◽

Protein Expression ◽

Mtt Assay ◽

Hepg2 Cells ◽

Molecular Mechanisms ◽

Migration And Invasion ◽

Rt Pcr ◽

Gene And Protein Expression ◽

Simulated Hypoxia

Background: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. Methods: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results： ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours’ relatively hypoxia incubation. Conclusion: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly

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Mass Spectrometric Calibration Procedure for Real-Time Detection of Lighter Hydrocarbons

Energies ◽

10.3390/en14082123 ◽

2021 ◽

Vol 14 (8) ◽

pp. 2123

Author(s):

Makuachukwu F. Mbaegbu ◽

Puspa L. Adhikari ◽

Ipsita Gupta ◽

Mathew Rowe

Keyword(s):

Real Time ◽

Mass Spectrometer ◽

Calibration Procedure ◽

Mass Spectrometric ◽

Spectrometric Method ◽

Mass Selection ◽

Mass Spectrometric Method ◽

Mass Spectrometers ◽

Drilling Rig

Determining gas compositions from live well fluids on a drilling rig is critical for real time formation evaluation. Development and utilization of a reliable mass spectrometric method to accurately characterize these live well fluids are always challenging due to lack of a robust and effectively selective instrument and procedure. The methods currently utilized need better calibration for the characterization of light hydrocarbons (C1–C6) at lower concentrations. The primary goal of this research is to develop and optimize a powerful and reliable analytical method to characterize live well fluid using a quadruple mass spectrometer (MS). The mass spectrometers currently being used in the field have issues with detection, spectra deconvolution, and quantification of analytes at lower concentrations (10–500 ppm), particularly for the lighter (<30 m/z) hydrocarbons. The objectives of the present study are thus to identify the detection issues, develop and optimize a better method, calibrate and QA/QC the MS, and validate the MS method in lab settings. In this study, we used two mass spectrometers to develop a selective and precise method to quantitatively analyze low level lighter analytes (C1–C6 hydrocarbons) with masses <75 m/z at concentrations 10–500 ppm. Our results suggest that proper mass selection like using base peaks with m/z 15, 26, 41, 43, 73, and 87, respectively, for methane, ethane, propane, butane, pentane, and hexane can help detect and accurately quantify hydrocarbons from gas streams. This optimized method in quadrupole mass spectrometer (QMS) will be invaluable for early characterization of the fluid components from a live hydrocarbon well in the field in real time.

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Development of a mass spectrometric method to quantitate platelet activating factor in mouse urine.

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)38209-2 ◽

1989 ◽

Vol 30 (12) ◽

pp. 1977-1981

Author(s):

E Benfenati ◽

D Macconi ◽

M Noris ◽

G Icardi ◽

L Bettazzoli ◽

...

Keyword(s):

Platelet Activating Factor ◽

Mass Spectrometric ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Mouse Urine

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Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of lenalidomide in human plasma and its application on bioequivalence studies

Journal of Analytical Science & Technology ◽

10.1186/s40543-019-0195-z ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

R. Gopinath ◽

S. T. Narenderan ◽

M. Kumar ◽

B. Babu

Keyword(s):

Liquid Chromatography ◽

Human Plasma ◽

Internal Standard ◽

Spectrometric Method ◽

Tandem Mass ◽

Mass Spectrometric Method ◽

Mass Detection ◽

Triple Quadrupole Mass Spectrometer ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometric

AbstractA simple, sensitive, and specific liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) method was developed and validated for the quantification of lenalidomide in human plasma. The separation was carried out on a symmetry, C18, 5-μm (50 × 4.6 mm) column as stationary phase and with an isocratic mobile phase of 0.1% formic acid in water-methanol in the ratio of (15:85, v/v) at a flow rate of 0.5 mL/min. Protonated ions formed by electrospray ionization in the positive mode were used to detect analyte and fluconazole (internal standard). The mass detection was made by monitoring the fragmentation of m/z 260.1/148.8 for lenalidomide and m/z 307.1/238.0 for internal standard on a triple quadrupole mass spectrometer. The developed method was validated over the concentration range of 10–1000 ng/mL for lenalidomide in human plasma with a correlation coefficient (r2) was 0.9930. The accuracy and precision values obtained from six different sets of quality control samples analyzed on separate occasions ranged from 99.41 to 106.97% and 2.88 to 4.22%, respectively. Mean extraction recoveries were 98.06% and 88.78% for the analyte and IS, respectively. The developed method was successfully applied for analyzing lenalidomide in human plasma samples.

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Molecular Mechanisms behind Inherited Neurodegeneration of the Optic Nerve

Biomolecules ◽

10.3390/biom11040496 ◽

2021 ◽

Vol 11 (4) ◽

pp. 496

Author(s):

Alessandra Maresca ◽

Valerio Carelli

Keyword(s):

Optic Nerve ◽

Mitochondrial Dysfunction ◽

Molecular Mechanisms ◽

Ganglion Cells ◽

Mitochondrial Dynamics ◽

Unmet Need ◽

Deep Understanding ◽

Genetic Landscape ◽

Innovative Therapies ◽

Energy Dependent

Inherited neurodegeneration of the optic nerve is a paradigm in neurology, as many forms of isolated or syndromic optic atrophy are encountered in clinical practice. The retinal ganglion cells originate the axons that form the optic nerve. They are particularly vulnerable to mitochondrial dysfunction, as they present a peculiar cellular architecture, with axons that are not myelinated for a long intra-retinal segment, thus, very energy dependent. The genetic landscape of causative mutations and genes greatly enlarged in the last decade, pointing to common pathways. These mostly imply mitochondrial dysfunction, which leads to a similar outcome in terms of neurodegeneration. We here critically review these pathways, which include (1) complex I-related oxidative phosphorylation (OXPHOS) dysfunction, (2) mitochondrial dynamics, and (3) endoplasmic reticulum-mitochondrial inter-organellar crosstalk. These major pathogenic mechanisms are in turn interconnected and represent the target for therapeutic strategies. Thus, their deep understanding is the basis to set and test new effective therapies, an urgent unmet need for these patients. New tools are now available to capture all interlinked mechanistic intricacies for the pathogenesis of optic nerve neurodegeneration, casting hope for innovative therapies to be rapidly transferred into the clinic and effectively cure inherited optic neuropathies.

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