bims-mascan 2021-01-10 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2021–01–10
thirty-two papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Metabolites and the tumour microenvironment: from cellular mechanisms to systemic metabolism.
Targeted reduction of cholesterol uptake in cholesterol-addicted lymphoma cells blocks turnover of oxidized lipids to cause ferroptosis.
The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.
Step-by-Step Sample Preparation of Proteins for Mass Spectrometric Analysis.
Mass spectrometry-based characterization of histones in clinical samples: applications, progresses and challenges.
Harnessing the Co-vulnerabilities of Amino Acid-Restricted Cancers.
The diversity and breadth of cancer cell fatty acid metabolism.
Phospholipid dynamics in ex vivo lung cancer and normal lung explants.
Ethical principles, opportunities and constraints in clinical proteomics.
Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum.
Remodeling Lipids in the Transition from Chronic Liver Disease to Hepatocellular Carcinoma.
Analytical Platforms for the Determination of Phospholipid Turnover in Breast Cancer Tissue: Role of Phospholipase Activity in Breast Cancer Development.
How Modern Mass Spectrometry Can Solve Ancient Questions: A Multi-Omics Study of the Stomach Content of the Oldest Human Ice Mummy, the 5300-Year-Old Iceman or Oetzi.
Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation.
Creation and Filtering of a Recurrent Spectral Library of CHO Cell Metabolites and Media Components.
Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study.
Metabolic Reprogramming of Cancer Cells during Tumor Progression and Metastasis.
Isolation and Purification of Mitochondria from Cell Culture for Proteomic Analyses.
Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue.
Isolation of Proteins from Extracellular Vesicles (EVs) for Mass Spectrometry-Based Proteomic Analyses.
Exploiting the metabolic dependencies of the broad amino acid transporter SLC6A14.
An Integrated Qual/Quan Strategy for Ganglioside Lipidomics utilizing High Resolution Mass Spectrometry and Skyline Software.
Reproducibility of Targeted Lipidome Analyses (Lipidyzer) in Plasma and Erythrocytes over a 6-Week Period.
Di-methylation of CD147-K234 Promotes the Progression of NSCLC by Enhancing Lactate Export.
Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis.
Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.
Stearoyl-CoA Desaturase-Mediated Monounsaturated Fatty Acid Availability Supports Humoral Immunity.
Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets.
Immunoaffinity-Based Liquid Chromatography Mass Spectrometric Assay to Accurately Quantify the Protein Concentration of HMGB1 in EDTA Plasma.
Deletion of Trim28 in committed adipocytes promotes obesity but preserves glucose tolerance.
MIEF2 reprograms lipid metabolism to drive progression of ovarian cancer through ROS/AKT/mTOR signaling pathway.
Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma.

Nat Metab. 2021 Jan 04.

Metabolites and the tumour microenvironment: from cellular mechanisms to systemic metabolism.

Ilaria Elia, Marcia C Haigis.

Metabolic transformation is a hallmark of cancer and a critical target for cancer therapy. Cancer metabolism and behaviour are regulated by cell-intrinsic factors as well as metabolite availability in the tumour microenvironment (TME). This metabolic niche within the TME is shaped by four tiers of regulation: (1) intrinsic tumour cell metabolism, (2) interactions between cancer cells and non-cancerous cells, (3) tumour location and heterogeneity and (4) whole-body metabolic homeostasis. Here, we define these modes of metabolic regulation and review how distinct cell types contribute to the metabolite composition of the TME. Finally, we connect these insights to understand how each of these tiers offers unique therapeutic potential to modulate the metabolic profile and function of all cells inhabiting the TME.

DOI: https://doi.org/10.1038/s42255-020-00317-z
J Biol Chem. 2020 Nov 24. pii: S0021-9258(20)00090-3. [Epub ahead of print]296 100100

Targeted reduction of cholesterol uptake in cholesterol-addicted lymphoma cells blocks turnover of oxidized lipids to cause ferroptosis.

Jonathan S Rink, Adam Yuh Lin, Kaylin M McMahon, Andrea E Calvert, Shuo Yang, Tim Taxter, Jonathan Moreira, Amy Chadburn, Amir Behdad, Reem Karmali, C Shad Thaxton, Leo I Gordon.

  Normal human cells can either synthesize cholesterol or take it up from lipoproteins to meet their metabolic requirements. In some malignant cells, de novo cholesterol synthesis genes are transcriptionally silent or mutated, meaning that cholesterol uptake from lipoproteins is required for survival. Recent data suggest that lymphoma cells dependent upon lipoprotein-mediated cholesterol uptake are also subject to ferroptosis, an oxygen- and iron-dependent cell death mechanism triggered by accumulation of oxidized lipids in cell membranes unless the lipid hydroperoxidase, glutathione peroxidase 4 (GPX4), reduces these toxic lipid species. To study mechanisms linking cholesterol uptake with ferroptosis and determine the potential role of the high-density lipoprotein (HDL) receptor as a target for cholesterol depleting therapy, we treated lymphoma cell lines known to be sensitive to the reduction of cholesterol uptake with HDL-like nanoparticles (HDL NPs). HDL NPs are a cholesterol-poor ligand that binds to the receptor for cholesterol-rich HDLs, scavenger receptor type B1 (SCARB1). Our data reveal that HDL NP treatment activates a compensatory metabolic response in treated cells toward increased de novo cholesterol synthesis, which is accompanied by nearly complete reduction in expression of GPX4. As a result, oxidized membrane lipids accumulate, leading to cell death through a mechanism consistent with ferroptosis. We obtained similar results in vivo after systemic administration of HDL NPs in mouse lymphoma xenografts and in primary samples obtained from patients with lymphoma. In summary, targeting SCARB1 with HDL NPs in cholesterol uptake-addicted lymphoma cells abolishes GPX4, resulting in cancer cell death by a mechanism consistent with ferroptosis.

Keywords:  cholesterol; ferroptosis; glutathione peroxidase 4 (GPX4); high-density lipoprotein (HDL); lipid peroxidation; lymphoma; nanotechnology; scavenger receptor type B1 (SCARB1)

DOI:  https://doi.org/10.1074/jbc.RA120.014888
Nat Genet. 2021 Jan;53(1): 16-26

The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.

CRUK Rosetta Grand Challenge Consortium

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

DOI: https://doi.org/10.1038/s41588-020-00753-3
Methods Mol Biol. 2021 ;2261 13-23

Step-by-Step Sample Preparation of Proteins for Mass Spectrometric Analysis.

Xinping Li, Thomas Franz, Ilian Atanassov, Thomas Colby.

  Nowadays identification and quantification of proteins from biological samples by mass spectrometry are widely used. For the identification of proteins, there are two scenarios. Proteins are either pre-fractionated in some way, e.g., by gel electrophoresis or chromatography, or analyzed as complex mixture (shotgun). Because of technological developments of mass spectrometry, the identification of several thousand proteins from complex biological matrix becomes possible. However, in many cases, it is still useful to separate proteins first in a gel. For quantifying proteins, label-free, isotopic labeling, and data-independent acquisition (DIA) library are widely used. Not only mass spectrometry technology made progress. This is also true for the sample preparation. Protocols and techniques developed recently not only make the analysis of starting material in the low microgram range possible but also simplify the whole procedure. Here, we will describe some detailed protocols of preparing samples for mass spectrometry-based protein identification and protein quantification, as in-gel digestion, in-solution digestion, peptide cleaning, and TMT labeling. This will allow also inexperienced beginners to get good results.

Keywords:  FASP; In-gel digest; In-solution digest; Mass spectrometry; OASIS; Proteomics; Sample preparation; StageTip; TMT labeling

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_2
FEBS J. 2021 Jan 08.

Mass spectrometry-based characterization of histones in clinical samples: applications, progresses and challenges.

Roberta Noberini, Giulia Robusti, Tiziana Bonaldi.

  In the last 15 years, increasing evidence linking epigenetics to various aspects of cancer biology has prompted the investigation of histone post-translational modifications (PTMs) and histone variants in the context of clinical samples. The studies performed so far demonstrated the potential of this type of investigations for the discovery of both potential epigenetic biomarkers for patient stratification and novel epigenetic mechanisms potentially targetable for cancer therapy. Although traditionally the analysis of histones in clinical samples was performed through antibody-based methods, mass spectrometry (MS) has emerged as a more powerful tool for the unbiased, comprehensive and quantitative investigation of histone PTMs and variants. MS has been extensively used for the analysis of epigenetic marks in cell lines and animal tissue, and, thanks to recent technological advances, is now ready to be applied also to clinical samples. In this review, we will provide an overview on the quantitative MS-based analysis of histones, their PTMs and their variants in cancer clinical samples, highlighting current achievements and future perspectives for this novel field of research. Among the different MS-based approaches currently available for histone PTM profiling, we will focus on the "bottom-up" strategy, namely the analysis of short proteolytic peptides, as it has been already successfully employed for the analysis clinical samples.

Keywords:  biomarker; cancer; chromatin; epigenetics; histone modifying enzyme; histone post-translational modification; histone variant; mass spectrometry; proteomics

DOI:  https://doi.org/10.1111/febs.15707
Cell Metab. 2021 Jan 05. pii: S1550-4131(20)30664-1. [Epub ahead of print]33(1): 9-20

Harnessing the Co-vulnerabilities of Amino Acid-Restricted Cancers.

Gaurav Pathria, Ze'ev A Ronai.

  Sustained proliferative potential of cancer cells creates heightened energetic and biosynthetic demands. The resulting overt dependence of cancer cells on unperturbed nutrient supply has prompted a widespread interest in amino acid restriction strategies as potential cancer therapeutics. However, owing to rapid signaling and metabolic reprogramming in cancer cells, the prospects for success of amino acid restriction approaches remain unclear. We thus recognize that the identification of co-vulnerabilities of amino acid-restricted cancers may inform actionable targets for effective combined interventions. In this perspective, we outline the current state of key cellular mechanisms underlying adaptation to amino acid restriction and discuss the role of signal transduction pathways governing cancer cell resistance to amino acid restriction, with potential ramifications for the design of future therapeutic efforts.

Keywords:  ATF4; MAPK; NRF2; adaptation; amino acids; c-MYC; cancer; mTORC1; metabolism; resistance

DOI:  https://doi.org/10.1016/j.cmet.2020.12.009
Cancer Metab. 2021 Jan 07. 9(1): 2

The diversity and breadth of cancer cell fatty acid metabolism.

Shilpa R Nagarajan, Lisa M Butler, Andrew J Hoy.

  Tumor cellular metabolism exhibits distinguishing features that collectively enhance biomass synthesis while maintaining redox balance and cellular homeostasis. These attributes reflect the complex interactions between cell-intrinsic factors such as genomic-transcriptomic regulation and cell-extrinsic influences, including growth factor and nutrient availability. Alongside glucose and amino acid metabolism, fatty acid metabolism supports tumorigenesis and disease progression through a range of processes including membrane biosynthesis, energy storage and production, and generation of signaling intermediates. Here, we highlight the complexity of cellular fatty acid metabolism in cancer, the various inputs and outputs of the intracellular free fatty acid pool, and the numerous ways that these pathways influence disease behavior.

Keywords:  Cellular membrane; De novo synthesis; Fatty acid; Lipid; Lipid droplets; Mitochondria; Oxidation; Peroxisome

DOI:  https://doi.org/10.1186/s40170-020-00237-2
Exp Mol Med. 2021 Jan 06.

Phospholipid dynamics in ex vivo lung cancer and normal lung explants.

Julia Lesko, Alexander Triebl, Elvira Stacher-Priehse, Nicole Fink-Neuböck, Jörg Lindenmann, Freyja-Maria Smolle-Jüttner, Harald C Köfeler, Andelko Hrzenjak, Horst Olschewski, Katharina Leithner.

In cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully 13C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.

DOI: https://doi.org/10.1038/s12276-020-00547-x
Mol Cell Proteomics. 2021 Jan 04. pii: mcp.RA120.002435. [Epub ahead of print]

Ethical principles, opportunities and constraints in clinical proteomics.

Sebastian Porsdam Mann, Peter V Treit, Philipp Emanuel Geyer, Gilbert S Omenn, Matthias Mann.

  Recent advances in MS-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with the impact on the health, privacy, and well-being of individuals. Here we conducted and report a systematic literature review of ethical issues in clinical proteomics. We add our perspectives from a background of bioethics, the results of our accompanying paper extracting individual-sensitive results from patient samples, and the literature addressing similar issues in genomics. The spectrum of potential issues ranges from patient re-identification to incidental findings of clinical significance. The latter can be divided into actionable and unactionable findings. Some of these have the potential to be employed in discriminatory or privacy-infringing ways. However, incidental findings may also have great positive potential. A plasma proteome profile, for instance, could inform on the general health or disease status of an individual regardless of the narrow diagnostic question that prompted it. We suggest that early discussion of ethical issues in clinical proteomics is important to ensure that eventual regulations reflect the considered judgment of the community as well as to anticipate opportunities and problems that may arise as the technology matures further.

Keywords:  Biomarker: Diagnostic; Biomarker: Prognostic; Clinical data; Clinical proteomics; Consent; Data evaluation; Diagnostic; Ethics; Human Rights; Incidental Findings; Individualized medicine*; Mass Spectrometry; Personalized medicine; Quality control and metrics; Regulations

DOI:  https://doi.org/10.1074/mcp.RA120.002435
Metabolites. 2020 Dec 30. pii: E19. [Epub ahead of print]11(1):

Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum.

Suzumi M Tokuoka, Yoshihiro Kita, Masaya Sato, Takao Shimizu, Yutaka Yatomi, Yoshiya Oda.

  In clinical lipidomics, it is a challenge to measure a large number of samples and to reproduce the quantitative results. We expanded the range of application of the tandem mass tag (TMT) method, which is widely used in proteomics, to lipidomic fields. There are various types of lipid molecule, for example, eicosanoids have a carboxyl group and phosphatidic acid has a phosphate group. We modified these functional groups simultaneously with TMT. This approach allows for a single analysis by mixing six samples and using one of the six samples as a bridging sample; the quantitative data can be easily normalized even if the number of measurements increases. To accommodate a large number of samples, we utilize a pooled serum sample of 300 individuals as a bridging sample. The stability of these lipid molecules in serum was examined as an analytical validation for the simultaneous TMT labeling. It was found that the stability of these lipid molecules in serum differs greatly depending on the lipid species. These findings reaffirmed the importance of proper sample preparation and storage to obtain reliable data. The TMT labeling method is expected to be a useful method for lipidomics with high-throughput and reliable reproducibility.

Keywords:  high-throughput; lipids with carboxy groups; lipids with phosphate groups; relative quantitation

DOI:  https://doi.org/10.3390/metabo11010019
Cancers (Basel). 2020 Dec 30. pii: E88. [Epub ahead of print]13(1):

Remodeling Lipids in the Transition from Chronic Liver Disease to Hepatocellular Carcinoma.

Israa T Ismail, Ashraf Elfert, Marwa Helal, Ibrahim Salama, Hala El-Said, Oliver Fiehn.

  Hepatocellular carcinoma (HCC) is a worldwide health problem. HCC patients show a 50% mortality within two years of diagnosis. To better understand the molecular pathogenesis at the level of lipid metabolism, untargeted UPLC MS-QTOF lipidomics data were acquired from resected human HCC tissues and their paired nontumor hepatic tissues (n = 46). Blood samples of the same HCC subjects (n = 23) were compared to chronic liver disease (CLD) (n = 15) and healthy control (n = 15) blood samples. The participants were recruited from the National Liver Institute in Egypt. The lipidomics data yielded 604 identified lipids that were divided into six super classes. Five-hundred and twenty-four blood lipids were found as significantly differentiated (p < 0.05 and qFDR p < 0.1) between the three study groups. In the blood of CLD patients compared to healthy control subjects, almost all lipid classes were significantly upregulated. In CLD patients, triacylglycerides were found as the most significantly upregulated lipid class at qFDR p = 1.3 × 10-56, followed by phosphatidylcholines at qFDR p = 3.3 × 10-51 and plasmalogens at qFDR p = 1.8 × 10-46. In contrast, almost all blood lipids were significantly downregulated in HCC patients compared to CLD patients, and in HCC tissues compared to nontumor hepatic tissues. Ceramides were found as the most significant lipid class (qFDR p = 1 × 10-14) followed by phosphatidylglycerols (qFDR p = 3 × 10-9), phosphatidylcholines and plasmalogens. Despite these major differences, there were also common trends in the transitions between healthy controls, CLD and HCC patients. In blood, several mostly saturated triacylglycerides showed a continued increase in the trajectory towards HCC, accompanied by reduced levels of saturated free fatty acids and saturated lysophospatidylcholines. In contrast, the largest overlaps of lipid alterations that were found in both HCC tissue and blood comparisons were decreased levels of phosphatidylglycerols and sphingolipids. This study highlights the specific impact of HCC tumors on the circulating lipids. Such data may be used to target lipid metabolism for prevention, early detection and treatment of HCC in the background of viral-related CLD etiology.

Keywords:  cirrhosis; desaturase; lipoproteins; palmitate; polyunsaturated fatty acids

DOI:  https://doi.org/10.3390/cancers13010088
Metabolites. 2021 Jan 04. pii: E32. [Epub ahead of print]11(1):

Analytical Platforms for the Determination of Phospholipid Turnover in Breast Cancer Tissue: Role of Phospholipase Activity in Breast Cancer Development.

Rosa Perestrelo, Marijana Petkovic, Catarina Luís Silva.

  Altered lipid metabolism has been associated with the progression of various cancers, and aberrant expression of enzymes involved in the lipid metabolism has been detected in different stages of cancer. Breast cancer (BC) is one of the cancer types known to be associated with alterations in the lipid metabolism and overexpression of enzymes involved in this metabolism. It has been demonstrated that inhibition of the activity of certain enzymes, such as that of phospholipase A2 in BC cell lines sensitizes these cells and decreases the IC50 values for forthcoming therapy with traditional drugs, such as doxorubicin and tamoxifen. Moreover, other phospholipases, such as phospholipase C and D, are involved in intracellular signal transduction, which emphasizes their importance in cancer development. Finally, BC is assumed to be dependent on the diet and the composition of lipids in nutrients. Despite their importance, analytical approaches that can associate the activity of phospholipases with changes in the lipid composition and distribution in cancer tissues are not yet standardized. In this review, an overview of various analytical platforms that are applied on the study of lipids and phospholipase activity in BC tissues will be given, as well as their association with cancer diagnosis and tumor progression. The methods that are applied to tissues obtained from the BC patients will be emphasized and critically evaluated, regarding their applicability in oncology.

Keywords:  analytical platforms; breast cancer; lipids; statistical analysis

DOI:  https://doi.org/10.3390/metabo11010032
Methods Mol Biol. 2021 ;2261 1-12

How Modern Mass Spectrometry Can Solve Ancient Questions: A Multi-Omics Study of the Stomach Content of the Oldest Human Ice Mummy, the 5300-Year-Old Iceman or Oetzi.

Rudolf Grimm.

  In the past 40 years, mass spectrometry has seen a stunning development regarding increased sensitivity, resolution, and accuracy, especially for biomolecule analysis. These days without any doubt mass spectrometry is the most powerful analytical tool as a standalone technique or in conjunction with separation techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC), or capillary electrophoresis (CE). It is literally used to analyze any kind of small or large molecules ranging from basic elements to metabolites, pesticides, toxins, small or large molecule drugs, oligonucleotides, peptides, proteins, and many other molecule classes.Here, various modern mass spectrometry techniques such as LC-MS , GC-MS, ICP-MS, and elemental bio-imaging are briefly described how they were used for the first complex multi-omics study of the oldest human ice mummy, the 5300-year-old Iceman or Oetzi. The study comprised of mass spectrometry-driven proteomics (protein profiling and characterization), metabolomics, lipidomics, glycomics, and metallomics.

Keywords:  Electrospray ionization (ESI) mass spectrometry; Elemental bio-imaging; Glycomics; Iceman; Inductively coupled plasma mass spectrometry (ICP-MS); Lipidomics; Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry; Metabolomics; Metallomics; Oetzi; Proteomics

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_1
Methods Mol Biol. 2021 ;2261 55-72

Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation.

Marcella Nunes Melo-Braga, María Ibáñez-Vea, Katarzyna Kulej, Martin R Larsen.

  Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

Keywords:  Acetylation; Comprising of titanium dioxide (TiO2); Hydrophilic interaction liquid chromatography (HILIC)/high pH reversed phase chromatography (HpH); Immunoprecipitation (IP); Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS); Phosphorylation; Protein posttranslational modification (PTM) enrichment; Sequential elution from immobilized metal affinity chromatography (SIMAC); Sialic acid (SA) N-linked glycosylation; TiSH

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_5
Biotechnol Bioeng. 2021 Jan 06.

Creation and Filtering of a Recurrent Spectral Library of CHO Cell Metabolites and Media Components.

Kelly H Telu, Ramesh Marupaka, Nirina R Andriamaharavo, Yamil Simón-Manso, Yuxue Liang, Yuri A Mirokhin, Tallat H Bukhari, Renae J Preston, Lila Kashi, Zvi Kelman, Stephen E Stein.

  This paper reports the first implementation of a new type of mass spectral library for the analysis of Chinese hamster ovary (CHO) cell metabolites that allows users to quickly identify most compounds in any complex metabolite sample. We also describe an annotation methodology developed to filter out artifacts and low-quality spectra from recurrent unidentified spectra of metabolites. CHO cells are commonly used to produce biological therapeutics. Metabolic profiles of CHO cells and media can be used to monitor process variability and look for markers that discriminate between batches of product. We have created a comprehensive library of both identified and unidentified metabolites derived from CHO cells that can be used in conjunction with tandem mass spectrometry to identify metabolites. In addition, we present a workflow that can be used for assigning confidence to a NIST MS/MS Library search match based on prior probability of general utility. The goal of our work is to annotate and identify (when possible), all liquid chromatography mass spectrometry (LC-MS) generated metabolite ions as well as create automatable library building and identification pipelines for use by others in the field. This article is protected by copyright. All rights reserved.

Keywords:  Chinese Hamster Ovary cells; Global Metabolite Profiling; Liquid Chromatography-Tandem Mass Spectrometry; Non-targeted Metabolomics; Recurrent Unidentified Spectra

DOI:  https://doi.org/10.1002/bit.27661
Anticancer Res. 2021 Jan;41(1): 327-334

Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study.

Hiroki Mori, Yuji Morine, Kazuaki Mawatari, Ayumi Chiba, Shinichiro Yamada, Y U Saito, Hiroki Ishibashi, Akira Takahashi, Mitsuo Shimada.

   BACKGROUND/AIM: Pancreaticobiliary maljunction (PBM), a disease with reflux of pancreatic and bile juice in the pancreaticobiliary tract, is a high-risk factor for biliary tract cancer. The aim of this study was to investigate the mechanism of carcinogenesis in PBM using a metabolomics analysis of bile sampled during surgery.
PATIENTS AND METHODS: Three patients with PBM without biliary tract cancer, four patients with extrahepatic bile duct cancer (EHBC), and three controls with benign disease were enrolled. Metabolomics analysis of bile samples was performed using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to discriminate the amino acid and lipidomic profiles.
RESULTS: The principal component analysis in the capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry revealed similar metabolites in patients with PBM and those with EHBC; furthermore, there was a clear difference between patients with PBM or EHBC compared to controls. The amino acid profiles revealed the following 20 potential carcinogenic candidates for PBM: isoleucine, phenylalanine, tyrosine, leucine, tryptophan, arginine, lysine, valine, asparagine, methionine, aspartic acid, serine, threonine, histidine, glutamine, alanine, proline, glutamic acid, and pyruvic acid. The lipidomic profiles revealed the following 11 carcinogenic candidates: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl glycerol, lysophosphatidyl glycerol, triacylglycerol, diacylglycerol, ceramide, sphyngomyeline, fatty acid, hyperforin, and vitamin D. Among these characteristic metabolites, the branched-chain amino acids, methionine and lysophosphatidylcholine are known to be related to carcinogenesis.
CONCLUSION: The bile metabolites were extremely similar in patients with PBM and those with EHBC. Furthermore, amino acid and lipid metabolism was markedly different in patients with PBM or EHBC compared to healthy controls.

Keywords:  Pancreaticobiliary maljunction; capillary electrophoresis-mass spectrometry (CE-MS); carcinogenesis; liquid chromatography-mass spectrometry (LC-MS); metabolomics

DOI:  https://doi.org/10.21873/anticanres.14779
Metabolites. 2021 Jan 02. pii: E28. [Epub ahead of print]11(1):

Metabolic Reprogramming of Cancer Cells during Tumor Progression and Metastasis.

Kenji Ohshima, Eiichi Morii.

  Cancer cells face various metabolic challenges during tumor progression, including growth in the nutrient-altered and oxygen-deficient microenvironment of the primary site, intravasation into vessels where anchorage-independent growth is required, and colonization of distant organs where the environment is distinct from that of the primary site. Thus, cancer cells must reprogram their metabolic state in every step of cancer progression. Metabolic reprogramming is now recognized as a hallmark of cancer cells and supports cancer growth. Elucidating the underlying mechanisms of metabolic reprogramming in cancer cells may help identifying cancer targets and treatment strategies. This review summarizes our current understanding of metabolic reprogramming during cancer progression and metastasis, including cancer cell adaptation to the tumor microenvironment, defense against oxidative stress during anchorage-independent growth in vessels, and metabolic reprogramming during metastasis.

Keywords:  anchorage-independent growth; cancer metabolism; cancer metastasis; metabolic reprograming; therapeutic strategy; tumor microenvironments

DOI:  https://doi.org/10.3390/metabo11010028
Methods Mol Biol. 2021 ;2261 411-419

Isolation and Purification of Mitochondria from Cell Culture for Proteomic Analyses.

Yaschar Kabiri, Christine von Toerne, Adriana Fontes, Percy A Knolle, Hans Zischka.

  In-depth analysis of the mitochondrial proteome can be greatly improved by analyzing isolated mitochondria instead of whole cells. However, isolation of sufficient amounts of mitochondria from cell culture has proven to be notoriously difficult due to small sample size. Thus, we have developed a reproducible, controllable, and highly customizable method to isolate high microgram to low milligram amounts of intact mitochondria from cell culture samples along with an optional density gradient purification. This chapter provides a methodological update of our approach and underlines the excellent quality and coverage of the mitochondrial proteome of crude and purified mitochondria from cultured liver cancer cell lines.

Keywords:  Balch homogenizer; Cell culture; Mitochondria; Proteomics

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_25
Methods Mol Biol. 2021 ;2261 525-533

Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue.

Omid Azimzadeh, Michael J Atkinson, Soile Tapio.

  Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.

Keywords:  Cross-linking; Formalin-fixed, paraffin-embedded (FFPE); Heart; Label-free; Protein extraction; Proteomics

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_33
Methods Mol Biol. 2021 ;2261 207-212

Isolation of Proteins from Extracellular Vesicles (EVs) for Mass Spectrometry-Based Proteomic Analyses.

Prabal Subedi, Michael Schneider, Michael J Atkinson, Soile Tapio.

  Extracellular vesicles (EVs) are freely circulating nano/micrometer-sized membrane-bound vesicles released by various cell types. Their cargo consists of proteins, lipids, metabolites, and different types of RNA molecules reflecting the origin of the secreting cell type or tissue. Since the EV cargo is constantly changing in response to pathological status or different environmental stressors, it has been extensively studied in the quest for biomarkers, especially in the cancer research. Mass spectrometry (MS)-based proteome analysis is a powerful tool to elucidate the protein cargo in EVs. This chapter describes and characterizes three MS-compatible lysis methods, namely by using urea, guanidium hydrochloride, and radioimmunoprecipitation buffer for isolating proteins from EVs.

Keywords:  Data-dependent acquisition; Exosomes; Extracellular vesicles (EVs); Mass spectrometry; Proteomics; Sample preparation

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_12
Oncotarget. 2020 Dec 01. 11(48): 4490-4503

Exploiting the metabolic dependencies of the broad amino acid transporter SLC6A14.

Francesca R Dejure, Joachim Butzer, Ralph K Lindemann, Balca R Mardin.

  Tumor cells typically enhance their metabolic capacity to sustain their higher rate of growth and proliferation. One way to elevate the nutrient intake into cancer cells is to increase the expression of genes encoding amino acid transporters, which may represent targetable vulnerabilities. Here, we study the regulation and function of the broad amino acid transporter SLC6A14 in combination with metabolic stress, providing insights into an uncharacterized aspect of the transporter activity. We analyze the pattern of transcriptional changes in a panel of breast cancer cell lines upon metabolic stress and found that SLC6A14 expression levels are increased in the absence of methionine. Methionine deprivation, which can be achieved via modulation of dietary methionine intake in tumor cells, in turn leads to a heightened activation of the AMP-activated kinase (AMPK) in SLC6A14-deficient cells. While SLC6A14 genetic deficiency does not have a major impact on cell proliferation, combined depletion of AMPK and SLC6A14 leads to an increase in apoptosis upon methionine starvation, suggesting that combined targeting of SLC6A14 and AMPK can be exploited as a therapeutic approach to starve tumor cells.

Keywords:  AMPK; SLC6A14; metabolic stress; methionine; transcriptional regulation

DOI:  https://doi.org/10.18632/oncotarget.27758
Rapid Commun Mass Spectrom. 2021 Jan 07. e9041

An Integrated Qual/Quan Strategy for Ganglioside Lipidomics utilizing High Resolution Mass Spectrometry and Skyline Software.

Asoka Ranasinghe, Eugene Ciccimaro, Celia D'Arienzo, Timothy V Olah, Paul Ponath, Serhiy Hnatyshyn.

RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood.
METHOD: The gangliosides from human blood, human plasma and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 μL/min) HPLC/high resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves utilizing commercially available gangliosides.
RESULTS: Protein precipitation resulted in ~60-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer.
CONCLUSIONS: A combination of simple extraction and Micro-HPLC/HRMS allowed for quantification of gangliosides in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.

DOI: https://doi.org/10.1002/rcm.9041
Metabolites. 2020 Dec 31. pii: E26. [Epub ahead of print]11(1):

Reproducibility of Targeted Lipidome Analyses (Lipidyzer) in Plasma and Erythrocytes over a 6-Week Period.

Marieke Loef, Johannes H von Hegedus, Mohan Ghorasaini, Féline P B Kroon, Martin Giera, Andreea Ioan-Facsinay, Margreet Kloppenburg.

  It is essential to measure lipid biomarkers with a high reproducibility to prevent biased results. We compared the lipid composition and inter-day reproducibility of lipid measurements in plasma and erythrocytes. Samples from 42 individuals (77% women, mean age 65 years, mean body mass index (BMI) 27 kg/m2), obtained non-fasted at baseline and after 6 weeks, were used for quantification of up to 1000 lipid species across 13 lipid classes with the Lipidyzer platform. Intraclass correlation coefficients (ICCs) were calculated to investigate the variability of lipid concentrations between timepoints. The ICC distribution of lipids in plasma and erythrocytes were compared using Wilcoxon tests. After data processing, the analyses included 630 lipids in plasma and 286 in erythrocytes. From these, 230 lipids overlapped between sample types. In plasma, 78% of lipid measurements were reproduced well to excellently, compared to 37% in erythrocytes. The ICC score distribution in plasma (median ICC 0.69) was significantly better than in erythrocytes (median ICC 0.51) (p-value < 0.001). At the class level, reproducibility in plasma was superior for triacylglycerols and cholesteryl esters while ceramides, diacylglycerols, (lyso)phosphatidylethanolamines, and sphingomyelins showed better reproducibility in erythrocytes. Although in plasma overall reproducibility was superior, differences at individual and class levels may favor the use of erythrocytes.

Keywords:  erythrocytes; lipids; metabolomics; plasma; reproducibility

DOI:  https://doi.org/10.3390/metabo11010026
Cell Metab. 2021 Jan 05. pii: S1550-4131(20)30665-3. [Epub ahead of print]33(1): 160-173.e6

Di-methylation of CD147-K234 Promotes the Progression of NSCLC by Enhancing Lactate Export.

Ke Wang, Wan Huang, Ruo Chen, Peng Lin, Tao Zhang, Yun-Feng Ni, Hao Li, Jiao Wu, Xiu-Xuan Sun, Jie-Jie Geng, Yu-Meng Zhu, Gang Nan, Wei Zhang, Xi Chen, Ping Zhu, Huijie Bian, Zhi-Nan Chen.

  CD147 is a tumor-associated glycoprotein that regulates cell metabolism. However, CD147 methylation and its subsequent role in cancer cell metabolism remain unclear. Here, we detect CD147 di-methylation in 16 non-small-cell lung cancer (NSCLC) tissues using liquid chromatography-tandem mass spectrometry. CD147 is di-methylated to CD147-K234me2 by lysine methyltransferase 5A (KMT5A). The increase in KMT5A expression boosts the levels of CD147-K234me2, further promoting the interaction between CD147 and monocarboxylate transporter 4 (MCT4), which enhances the translocation of MCT4 from the cytoplasm to the membrane. Overexpression of CD147-K234me2 and KMT5A enhances glycolysis and lactate export in NSCLC cells. Clinical analysis shows that high CD147-K234me2 expression is significantly related to cancer progression and overall survival, and has prognostic significance in individuals with NSCLC, especially for those in the early stages. Our findings indicate that CD147-K234me2 plays a critical role in cancer metabolism, and it can be a highly promising therapeutic target for NSCLC.

Keywords:  CD147; KMT5A; MCT4; NSCLC; di-methylation; lactate

DOI:  https://doi.org/10.1016/j.cmet.2020.12.010
Cell Death Dis. 2021 Jan 07. 12(1): 26

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis.

Faisal Thayyullathil, Anees Rahman Cheratta, Ameer Alakkal, Karthikeyan Subburayan, Siraj Pallichankandy, Yusuf A Hannun, Sehamuddin Galadari.

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.

DOI: https://doi.org/10.1038/s41419-020-03297-w
Theranostics. 2021 ;11(3): 1377-1395

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Raimund Dietze, Mohamad K Hammoud, María Gómez-Serrano, Annika Unger, Tim Bieringer, Florian Finkernagel, Anna M Sokol, Andrea Nist, Thorsten Stiewe, Silke Reinartz, Viviane Ponath, Christian Preußer, Elke Pogge von Strandmann, Sabine Müller-Brüsselbach, Johannes Graumann, Rolf Müller.

  Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

Keywords:  arachidonic acid; macropinocytosis; ovarian cancer; phosphoproteomics

DOI:  https://doi.org/10.7150/thno.52442
Cell Rep. 2021 Jan 05. pii: S2211-1247(20)31590-4. [Epub ahead of print]34(1): 108601

Stearoyl-CoA Desaturase-Mediated Monounsaturated Fatty Acid Availability Supports Humoral Immunity.

Xian Zhou, Xingxing Zhu, Chaofan Li, Yanfeng Li, Zhenqing Ye, Virginia Smith Shapiro, John A Copland, Taro Hitosugi, David A Bernlohr, Jie Sun, Hu Zeng.

  Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The roles of different FA species and their impacts on humoral immunity remain poorly understood. Here, we report that proliferating B cells require monounsaturated FAs (MUFAs) to maintain mitochondrial metabolism and mTOR activity and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Furthermore, B cell-extrinsic stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity.

Keywords:  B cell; autophagy; humoral immunity; mTOR; monounsaturated fatty acid; stearoyl-CoA desaturase

DOI:  https://doi.org/10.1016/j.celrep.2020.108601
Methods Mol Biol. 2021 ;2261 229-246

Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets.

Penny Jensen, Bhavin Patel, Suzanne Smith, Renuka Sabnis, Barbara Kaboord.

  Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss an improved IP-MS workflow that is designed to minimize sample prep time and these contaminants. The method employs biotinylated antibodies to the targets of interest and streptavidin magnetic beads that exhibit low background binding. In addition, alterations in the elution protocol and subsequent MS sample prep were made to reduce time and antibody leaching in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of multiple target antigens and interacting proteins.

Keywords:  Biotin; Co-immunoprecipitation; Immunoprecipitation; In-solution digestion; Magnetic beads; Mass spectrometry; On-bead digestion; Protein–protein interactions; Streptavidin

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_14
Methods Mol Biol. 2021 ;2261 277-289

Immunoaffinity-Based Liquid Chromatography Mass Spectrometric Assay to Accurately Quantify the Protein Concentration of HMGB1 in EDTA Plasma.

Viktoria Anselm, Andreas Steinhilber, Cornelia Sommersdorf, Oliver Poetz.

  Targeted protein quantification can be challenging in body fluids such as plasma with regard to sensitivity and selectivity. In this chapter, we present a protocol for the quantification of high mobility group box 1 protein (HMGB1) in plasma using an immunoaffinity liquid chromatography mass spectrometric assay (IA-LC-MSMS). The protocol provides detailed assay instructions involving sample proteolysis, peptide-targeted immunoprecipitation, and LC-MSMS-based read out.

Keywords:  Antibody; HMGB1; Immunoaffinity liquid chromatography mass spectrometric assay; Immunoprecipitation; Parallel reaction monitoring; Peptide quantification

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_17
Nat Commun. 2021 01 04. 12(1): 74

Deletion of Trim28 in committed adipocytes promotes obesity but preserves glucose tolerance.

Simon T Bond, Emily J King, Darren C Henstridge, Adrian Tran, Sarah C Moody, Christine Yang, Yingying Liu, Natalie A Mellett, Artika P Nath, Michael Inouye, Elizabeth J Tarling, Thomas Q de Aguiar Vallim, Peter J Meikle, Anna C Calkin, Brian G Drew.

The effective storage of lipids in white adipose tissue (WAT) critically impacts whole body energy homeostasis. Many genes have been implicated in WAT lipid metabolism, including tripartite motif containing 28 (Trim28), a gene proposed to primarily influence adiposity via epigenetic mechanisms in embryonic development. However, in the current study we demonstrate that mice with deletion of Trim28 specifically in committed adipocytes, also develop obesity similar to global Trim28 deletion models, highlighting a post-developmental role for Trim28. These effects were exacerbated in female mice, contributing to the growing notion that Trim28 is a sex-specific regulator of obesity. Mechanistically, this phenotype involves alterations in lipolysis and triglyceride metabolism, explained in part by loss of Klf14 expression, a gene previously demonstrated to modulate adipocyte size and body composition in a sex-specific manner. Thus, these findings provide evidence that Trim28 is a bona fide, sex specific regulator of post-developmental adiposity and WAT function.

DOI: https://doi.org/10.1038/s41467-020-20434-3
Cell Death Dis. 2021 Jan 05. 12(1): 18

MIEF2 reprograms lipid metabolism to drive progression of ovarian cancer through ROS/AKT/mTOR signaling pathway.

Shuhua Zhao, Lu Cheng, Yuan Shi, Jia Li, Qinghui Yun, Hong Yang.

MIEF2 (mitochondrial elongation factor 2) is one of the key regulators of mitochondrial fission. Bioinformatics analysis indicated that high expression of MIEF2 predicted a poor prognosis in ovarian cancer patients. However, the relationship between MIEF2 and aberrant lipid metabolism in OC remains elusive. In this study, we demonstrated that MIEF2 significantly promoted lipid synthesis, while has no significant effect on fatty acid uptake and oxidation in OC cells. MIEF2 enhanced de novo fatty acid synthesis through up-regulating the expression of sterol regulatory element binding protein 1 (SREBP1) and its transcriptional target lipogenic genes ACC1, FASN and SCD1. Meanwhile, MIEF2-promoted cholesterol biosynthesis through up-regulating the expression of sterol regulatory element binding protein 2 (SREBP2) and its transcriptional target cholesterol biosynthesis genes HMGCS1 and HMGCR. Mechanistically, increased mitochondrial reactive oxygen species (ROS) production and subsequently activation of AKT/mTOR signaling pathway was found to be involved in the up-regulation of SREBP1 and SREBP2 in OC cells. Moreover, cell growth and metastasis assays indicated that MIEF2-regulated fatty acid synthesis and cholesterol biosynthesis played a critical role in the progression of OC. Taken together, our findings indicate that MIEF2 is a critical regulator of lipid synthesis in OC, which provides a strong line of evidence for this molecule to serve as a drug target in the treatment of this malignancy.

DOI: https://doi.org/10.1038/s41419-020-03336-6
Methods Mol Biol. 2021 ;2261 151-191

Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma.

Antonia Reale, Tiffany Khong, Rong Xu, Maoshan Chen, Sridurga Mithraprabhu, Nicholas Bingham, Andrew Spencer, David W Greening.

  Cancer cells secrete membranous extracellular vesicles (EVs) which contain specific oncogenic molecular cargo (including oncoproteins, oncopeptides, and RNA) into their microenvironment and the circulation. As such, EVs including exosomes (small EVs) and microvesicles (large EVs) represent important circulating biomarkers for various diseases, including cancer and its progression. These circulating biomarkers offer a potentially minimally invasive and repeatable targets for analysis (liquid biopsy) that could aid in the diagnosis, risk stratification, and monitoring of cancer. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remain challenging. Like EVs, other types of circulating biomarkers (including cell-free nucleic acids, cf-NAs; or circulating tumor cells, CTCs) may represent a complementary or alternative approach to cancer diagnosis. In the context of multiple myeloma (MM), a systemic cancer type that causes cancer cells to accumulate in the bone marrow, the specific role for EVs as biomarkers for diagnosis and monitoring remains undefined. Tumor heterogeneity along with the various subtypes of MM (such as non-secretory MM) that cannot be monitored using conventional testing (e.g. sequential serological testing and bone marrow biopsies) render liquid biopsy and circulating tumor-derived EVs a promising approach. In this protocol, we describe the isolation and purification of EVs from peripheral blood plasma (PBPL) collected from healthy donors and patients with MM for a biomarker discovery strategy. Our results demonstrate detection of circulating EVs from as little as 1 mL of MM patients' PBPL. High-resolution mass spectrometry (MS)-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease. We describe biophysical characterization and quantitative proteomic profiling of disease-specific circulating EVs which may provide important implications for the development of cancer diagnostics in MM.

Keywords:  Blood; Exosomes; Extracellular vesicles; Liquid biopsy; Multiple myeloma

DOI:  https://doi.org/10.1007/978-1-0716-1186-9_10