Phosphoproteomics-directed manipulation reveals SEC22B as a hepatocellular signaling node governing metabolic actions of glucagon.

Animals Signal Transduction Glucagon / metabolism Phosphorylation Proteomics / methods Hepatocytes / metabolism Mice Liver / metabolism Vesicular Transport Proteins / metabolism Phosphoproteins / metabolism Male Mice, Inbred C57BL Humans Lipid Metabolism Glycogen / metabolism

Journal

Nature communications

ISSN: 2041-1723

Titre abrégé: Nat Commun

Pays: England

ID NLM: 101528555

Informations de publication

Date de publication:
27 Sep 2024

Historique:

received: 17 01 2024

accepted: 16 09 2024

medline: 28 9 2024

pubmed: 28 9 2024

entrez: 27 9 2024

Statut: epublish

Résumé

The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal "membrane trafficking" as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action.

Identifiants

DOI: 10.1038/s41467-024-52703-w PMID: 39333498

pubmed: 39333498

doi: 10.1038/s41467-024-52703-w

pii: 10.1038/s41467-024-52703-w

doi:

Substances chimiques

Glucagon 9007-92-5

Vesicular Transport Proteins 0

Phosphoproteins 0

Glycogen 9005-79-2

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

Pagination

8390

Subventions

Organisme : Diabetes Australia Research Trust (DART)

ID : Y22G

Informations de copyright

Références

Kimball C. P. & Murlin J. R. Aqueous extracts of pancreas: III. Some precipitation reactions of insulin. J. Biol. Chem. 58, (1923).

Finan, B., Capozzi, M. E. & Campbell, J. E. Repositioning glucagon action in the physiology and pharmacology of diabetes. Diabetes 69, 532–541 (2020).

pubmed: 31178432 doi: 10.2337/dbi19-0004

Lee, Y. H., Wang, M. Y., Yu, X. X. & Unger, R. H. Glucagon is the key factor in the development of diabetes. Diabetologia 59, 1372–1375 (2016).

pubmed: 27115412 doi: 10.1007/s00125-016-3965-9

Wewer Albrechtsen, N. J. Glucagon receptor signaling in metabolic diseases. Peptides 100, 42–47 (2018).

pubmed: 29412830 doi: 10.1016/j.peptides.2017.11.016

Müller, T. D., Finan, B., Clemmensen, C., DiMarchi, R. D. & Tschöp, M. H. The new biology and pharmacology of glucagon. Physiol. Rev. 97, 721–766 (2017).

pubmed: 28275047 doi: 10.1152/physrev.00025.2016

Ang, T., Bruce, C. R. & Kowalski, G. M. Postprandial aminogenic insulin and glucagon secretion can stimulate glucose flux in humans. Diabetes 68, 939–946 (2019).

pubmed: 30833465 doi: 10.2337/db18-1138

El, K., Capozzi, M. E. & Campbell, J. E. Repositioning the alpha cell in postprandial metabolism. Endocrinology 161, bqaa169 (2020).

pubmed: 32964214 pmcid: 7899437 doi: 10.1210/endocr/bqaa169

Day, J. W. et al. A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nat. Chem. Biol. 5, 749–757 (2009).

pubmed: 19597507 doi: 10.1038/nchembio.209

Boland, M. L. et al. Resolution of NASH and hepatic fibrosis by the GLP-1R/GcgR dual-agonist Cotadutide via modulating mitochondrial function and lipogenesis. Nat. Metab 2, 413–431 (2020).

pubmed: 32478287 pmcid: 7258337 doi: 10.1038/s42255-020-0209-6

Coskun, T. et al. LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept. Cell Metab 34, 1234–1247.e1239 (2022).

pubmed: 35985340 doi: 10.1016/j.cmet.2022.07.013

Finan, B. et al. A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nature Medicine 21, 27–36 (2015).

pubmed: 25485909 doi: 10.1038/nm.3761

Rosenstock, J. et al. Retatrutide, a GIP, GLP-1 and glucagon receptor agonist, for people with type 2 diabetes: a randomised, double-blind, placebo and active-controlled, parallel-group, phase 2 trial conducted in the USA. Lancet 402, 529–544 (2023).

pubmed: 37385280 doi: 10.1016/S0140-6736(23)01053-X

Jastreboff, A. M. et al. Triple-hormone-receptor agonist retatrutide for obesity - a phase 2 trial. N. Engl. J. Med. 389, 514–526 (2023).

pubmed: 37366315 doi: 10.1056/NEJMoa2301972

Goldstein, I. & Hager, G. L. The three Ds of transcription activation by glucagon: direct, delayed, and dynamic. Endocrinology 159, 206–216 (2018).

pubmed: 29077799 doi: 10.1210/en.2017-00521

Cohen, P. The role of protein phosphorylation in human health and disease. the Sir Hans Krebs Medal lecture. Eur. J. Biochem. 268, 5001–5010 (2001).

pubmed: 11589691 doi: 10.1046/j.0014-2956.2001.02473.x

Humphrey, S. J., James, D. E. & Mann, M. Protein phosphorylation: a major switch mechanism for metabolic regulation. Trends Endocrinol Metab 26, 676–687 (2015).

pubmed: 26498855 doi: 10.1016/j.tem.2015.09.013

Longuet, C. et al. Liver-specific disruption of the murine glucagon receptor produces α-cell hyperplasia: evidence for a circulating α-cell growth factor. Diabetes 62, 1196–1205 (2013).

pubmed: 23160527 pmcid: 3609565 doi: 10.2337/db11-1605

Ramnanan, C. J., Edgerton, D. S., Kraft, G. & Cherrington, A. D. Physiologic action of glucagon on liver glucose metabolism. Diabetes Obes Metab 13, 118–125 (2011).

pubmed: 21824265 pmcid: 5371022 doi: 10.1111/j.1463-1326.2011.01454.x

Johnson, J. L. et al. An atlas of substrate specificities for the human serine/threonine kinome. Nature 613, 759–766 (2023).

pubmed: 36631611 pmcid: 9876800 doi: 10.1038/s41586-022-05575-3

Wilson-Grady, J. T., Haas, W. & Gygi, S. P. Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 61, 277–286 (2013).

pubmed: 23567750 doi: 10.1016/j.ymeth.2013.03.031

Imamura, H. et al. Identifications of putative PKA substrates with quantitative phosphoproteomics and primary-sequence-based scoring. J Proteome Res 16, 1825–1830 (2017).

pubmed: 28287266 doi: 10.1021/acs.jproteome.7b00087

Isobe, K. et al. Systems-level identification of PKA-dependent signaling in epithelial cells. Proc. Natl Acad Sci. USA 114, E8875–e8884 (2017).

pubmed: 28973931 pmcid: 5651769 doi: 10.1073/pnas.1709123114

Marliss, E. B., Aoki, T. T., Unger, R. H., Soeldner, J. S. & Cahill, G. F. Jr Glucagon levels and metabolic effects in fasting man. J. Clin. Invest 49, 2256–2270 (1970).

pubmed: 5480852 pmcid: 322727 doi: 10.1172/JCI106445

Capozzi, M. E. et al. Glucagon lowers glycemia when β-cells are active. JCI Insight 5, e129954 (2019).

pubmed: 31335319 doi: 10.1172/jci.insight.129954

Fuhrmeister, J. et al. Fasting-induced liver GADD45β restrains hepatic fatty acid uptake and improves metabolic health. EMBO Mol. Med. 8, 654–669 (2016).

pubmed: 27137487 pmcid: 4888855 doi: 10.15252/emmm.201505801

Solloway, M. J. et al. Glucagon couples hepatic amino acid catabolism to mTOR-dependent regulation of α-cell mass. Cell Rep. 12, 495–510 (2015).

pubmed: 26166562 doi: 10.1016/j.celrep.2015.06.034

Sekar, R. et al. Vps37a regulates hepatic glucose production by controlling glucagon receptor localization to endosomes. Cell Metab 34, 1824–1842.e1829 (2022).

pubmed: 36243006 doi: 10.1016/j.cmet.2022.09.022

Habegger, K. M. et al. Fibroblast growth factor 21 mediates specific glucagon actions. Diabetes 62, 1453–1463 (2013).

pubmed: 23305646 pmcid: 3636653 doi: 10.2337/db12-1116

Pearlman, S. M., Serber, Z. & Ferrell, J. E. Jr. A mechanism for the evolution of phosphorylation sites. Cell 147, 934–946 (2011).

pubmed: 22078888 pmcid: 3220604 doi: 10.1016/j.cell.2011.08.052

Okun, J. G. et al. Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes. Nat. Metab 3, 394–409 (2021).

pubmed: 33758419 doi: 10.1038/s42255-021-00369-9

Han, S. et al. Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice. J. Lipid Res. 54, 2615–2622 (2013).

pubmed: 23828778 pmcid: 3770075 doi: 10.1194/jlr.M035592

Longuet, C. et al. The glucagon receptor is required for the adaptive metabolic response to fasting. Cell Metab 8, 359–371 (2008).

pubmed: 19046568 pmcid: 2593715 doi: 10.1016/j.cmet.2008.09.008

Perry, R. J. et al. Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis. Nature 579, 279–283 (2020).

pubmed: 32132708 pmcid: 7101062 doi: 10.1038/s41586-020-2074-6

Liang, Y. et al. Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice. Diabetes 53, 410–417 (2004).

pubmed: 14747292 doi: 10.2337/diabetes.53.2.410

Choi, J. H. et al. SEC22B inhibition attenuates colorectal cancer aggressiveness and autophagic flux under unfavorable environment. Biochem. Biophys. Res. Commun. 665, 10–18 (2023).

pubmed: 37148741 doi: 10.1016/j.bbrc.2023.03.039

Kimura, T. et al. Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy. Embo j 36, 42–60 (2017).

pubmed: 27932448 doi: 10.15252/embj.201695081

Siddiqi, S., Mani, A. M. & Siddiqi, S. A. The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi. Biochem. J 429, 391–401 (2010).

pubmed: 20450495 doi: 10.1042/BJ20100336

Karampini, E. et al. Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport. Haematologica 106, 1138–1147 (2021).

pubmed: 32336681 doi: 10.3324/haematol.2019.242727

Fan, J. et al. cTAGE5 deletion in pancreatic β cells impairs proinsulin trafficking and insulin biogenesis in mice. J. Cell Biol. 216, 4153–4164 (2017).

pubmed: 29133483 pmcid: 5716288 doi: 10.1083/jcb.201705027

Petkovic, M. et al. The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion. Nat. Cell Biol. 16, 434–444 (2014).

pubmed: 24705552 doi: 10.1038/ncb2937

Wu, S. J. et al. SNARE protein SEC22B regulates early embryonic development. Sci. Rep. 9, 11434 (2019).

pubmed: 31391476 pmcid: 6685974 doi: 10.1038/s41598-019-46536-7

Ge, L., Zhang, M. & Schekman, R. Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment. Elife 3, e04135 (2014).

pubmed: 25432021 pmcid: 4270069 doi: 10.7554/eLife.04135

Laidlaw, K. M. E., Livingstone, R., Al-Tobi, M., Bryant, N. J. & Gould, G. W. SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events. Biochem. Soc. Trans. 45, 1271–1277 (2017).

pubmed: 29101310 doi: 10.1042/BST20170202

Warner, H., Mahajan, S. & van den Bogaart, G. Rerouting trafficking circuits through posttranslational SNARE modifications. J. Cell Sci. 135, jcs260112 (2022).

pubmed: 35972760 doi: 10.1242/jcs.260112

Weinberger A. & Gerst J. E. Regulation of SNARE assembly by protein phosphorylation. In: Regulatory Mechanisms of Intracellular Membrane Transport). Springer (2004).

Sun, W., Tian, B. X., Wang, S. H., Liu, P. J. & Wang, Y. C. The function of SEC22B and its role in human diseases. Cytoskeleton (Hoboken) 77, 303–312 (2020).

pubmed: 32748571 doi: 10.1002/cm.21628

Turriziani, B., von Kriegsheim, A. & Pennington, S. R. Protein-protein interaction detection via mass spectrometry-based proteomics. Adv. Exp. Med. Biol. 919, 383–396 (2016).

pubmed: 27975227 doi: 10.1007/978-3-319-41448-5_18

Zeigerer, A. et al. Regulation of liver metabolism by the endosomal GTPase Rab5. Cell Rep 11, 884–892 (2015).

pubmed: 25937276 doi: 10.1016/j.celrep.2015.04.018

Seitz, S. et al. Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity. Nat. Metab 1, 1009–1026 (2019).

pubmed: 32694843 doi: 10.1038/s42255-019-0124-x

Yi Lo, J. C. et al. RABL2 is required for hepatic fatty acid homeostasis and its dysfunction leads to steatosis and a diabetes-like state. Endocrinology 157, 4732–4743 (2016).

pubmed: 27732084 doi: 10.1210/en.2016-1487

Hayashi, Y. & Seino, Y. Regulation of amino acid metabolism and α-cell proliferation by glucagon. J Diabetes Investig 9, 464–472 (2018).

pubmed: 29314731 pmcid: 5934249 doi: 10.1111/jdi.12797

Elmelund, E. et al. Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells. iScience 25, 105296 (2022).

pubmed: 36325048 pmcid: 9618771 doi: 10.1016/j.isci.2022.105296

Flakoll, P. J. et al. The role of glucagon in the control of protein and amino acid metabolism in vivo. Metabolism 43, 1509–1516 (1994).

pubmed: 7990704 doi: 10.1016/0026-0495(94)90009-4

Kim, J. et al. Amino acid transporter Slc38a5 controls glucagon receptor inhibition-induced pancreatic α cell hyperplasia in mice. Cell Metab 25, 1348–1361.e1348 (2017).

pubmed: 28591637 pmcid: 8206958 doi: 10.1016/j.cmet.2017.05.006

Dean, E. D. et al. Interrupted glucagon signaling reveals hepatic α cell axis and role for l-glutamine in α cell proliferation. Cell Metab 25, 1362–1373.e1365 (2017).

pubmed: 28591638 pmcid: 5572896 doi: 10.1016/j.cmet.2017.05.011

Madrigal-Matute, J. & Cuervo, A. M. Regulation of liver metabolism by autophagy. Gastroenterology 150, 328–339 (2016).

pubmed: 26453774 doi: 10.1053/j.gastro.2015.09.042

Jeon, Y. J. et al. Regulation of glutamine carrier proteins by RNF5 determines breast cancer response to ER stress-inducing chemotherapies. Cancer Cell 27, 354–369 (2015).

pubmed: 25759021 pmcid: 4356903 doi: 10.1016/j.ccell.2015.02.006

Paulusma, C. C., Lamers, W. H., Broer, S. & van de Graaf, S. F. J. Amino acid metabolism, transport and signalling in the liver revisited. Biochem. Pharmacol. 201, 115074 (2022).

pubmed: 35568239 doi: 10.1016/j.bcp.2022.115074

Hope, D. C. D. et al. Hypoaminoacidemia underpins glucagon-mediated energy expenditure and weight loss. Cell Rep. Med. 3, 100810 (2022).

pubmed: 36384093 pmcid: 9729826 doi: 10.1016/j.xcrm.2022.100810

Yap, Y. W. et al. Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution. Nat. Commun. 11, 2894 (2020).

pubmed: 32518324 pmcid: 7283339 doi: 10.1038/s41467-020-16568-z

Hinds, C. E. et al. A glucagon analogue decreases body weight in mice via signalling in the liver. Sci. Rep. 11, 22577 (2021).

pubmed: 34799628 pmcid: 8604983 doi: 10.1038/s41598-021-01912-0

Spolitu, S. et al. Hepatic glucagon signaling regulates PCSK9 and low-density lipoprotein cholesterol. Circ. Res. 124, 38–51 (2019).

pubmed: 30582457 pmcid: 6457439 doi: 10.1161/CIRCRESAHA.118.313648

Borén, J., Taskinen, M. R., Björnson, E. & Packard, C. J. Metabolism of triglyceride-rich lipoproteins in health and dyslipidaemia. Nat. Rev. Cardiol 19, 577–592 (2022).

pubmed: 35318466 doi: 10.1038/s41569-022-00676-y

Rahim, A. et al. Proteomic analysis of the very low density lipoprotein (VLDL) transport vesicles. J Proteomics 75, 2225–2235 (2012).

pubmed: 22449872 pmcid: 3341533 doi: 10.1016/j.jprot.2012.01.026

Tiwari, S. & Siddiqi, S. A. Intracellular trafficking and secretion of VLDL. Arterioscler Thromb Vasc Biol. 32, 1079–1086 (2012).

pubmed: 22517366 pmcid: 3334296 doi: 10.1161/ATVBAHA.111.241471

Fu, Y. et al. Qa-SNARE syntaxin 18 mediates lipid droplet fusion with SNAP23 and SEC22B. Cell Discov. 9, 115 (2023).

pubmed: 37989733 pmcid: 10663520 doi: 10.1038/s41421-023-00613-4

Huggett, Z. J. et al. A comparison of primary human hepatocytes and hepatoma cell lines to model the effects of fatty acids, fructose and glucose on liver cell lipid accumulation. Nutrients 15, 40 (2022).

pubmed: 36615698 pmcid: 9824391 doi: 10.3390/nu15010040

Jou, J., Choi, S. S. & Diehl, A. M. Mechanisms of disease progression in nonalcoholic fatty liver disease. Semin Liver Dis. 28, 370–379 (2008).

pubmed: 18956293 doi: 10.1055/s-0028-1091981

Zhang, X. et al. N6-methyladenosine modification governs liver glycogenesis by stabilizing the glycogen synthase 2 mRNA. Nat. Commun. 13, 7038 (2022).

pubmed: 36396934 pmcid: 9671881 doi: 10.1038/s41467-022-34808-2

Sun, T., Yi, H., Yang, C., Kishnani, P. S. & Sun, B. Starch binding domain-containing protein 1 plays a dominant role in glycogen transport to lysosomes in liver. J. Biol. Chem. 291, 16479–16484 (2016).

pubmed: 27358407 pmcid: 4974364 doi: 10.1074/jbc.C116.741397

Figlia, G., Willnow, P. & Teleman, A. A. Metabolites regulate cell signaling and growth via covalent modification of proteins. Dev. Cell 54, 156–170 (2020).

pubmed: 32693055 doi: 10.1016/j.devcel.2020.06.036

Graham, T., McIntosh, J., Work, L. M., Nathwani, A. & Baker, A. H. Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats. Genetic vaccines and therapy 6, 9 (2008).

pubmed: 18312698 pmcid: 2267784 doi: 10.1186/1479-0556-6-9

Rose, A. J. et al. Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor. Cell Metab 14, 123–130 (2011).

pubmed: 21723510 doi: 10.1016/j.cmet.2011.04.010

Jungmann, A., Leuchs, B., Rommelaere, J., Katus, H. A. & Müller, O. J. Protocol for efficient generation and characterization of adeno-associated viral vectors. Hum. Gene. Ther. Methods 28, 235–246 (2017).

pubmed: 29048971 doi: 10.1089/hgtb.2017.192

Gores, G. J., Kost, L. J. & LaRusso, N. F. The isolated perfused rat liver: conceptual and practical considerations. Hepatology 6, 511–517 (1986).

pubmed: 3519420 doi: 10.1002/hep.1840060331

Cheung, K. et al. An optimized model for rat liver perfusion studies. J. Surg. Res. 66, 81–89 (1996).

pubmed: 8954836 doi: 10.1006/jsre.1996.0376

Grundmann, M. & Kostenis, E. Temporal bias: time-encoded dynamic GPCR signaling. Trends Pharmacol. Sci. 38, 1110–1124 (2017).

pubmed: 29074251 doi: 10.1016/j.tips.2017.09.004

Hanein, S. et al. TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane. Biochim Biophys Acta 1830, 3719–3733 (2013).

pubmed: 23500070 doi: 10.1016/j.bbagen.2013.02.025

Formosa, L. E. et al. Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly. Proc Natl Acad Sci. USA 119, e2110357119 (2022).

pubmed: 35210360 pmcid: 8892353 doi: 10.1073/pnas.2110357119

Bruderer, R. et al. Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Mol Cell Proteomics 14, 1400–1410 (2015).

pubmed: 25724911 pmcid: 4424408 doi: 10.1074/mcp.M114.044305

Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

pubmed: 27348712 doi: 10.1038/nmeth.3901

Pang, Z. et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res. 49, W388–w396 (2021).

pubmed: 34019663 pmcid: 8265181 doi: 10.1093/nar/gkab382

Kolberg, L. et al. g:Profiler-interoperable web service for functional enrichment analysis and gene identifier mapping (2023 update). Nucleic Acids Res. 51, W207–w212 (2023).

pubmed: 37144459 pmcid: 10320099 doi: 10.1093/nar/gkad347

Oliveros J. C. VENNY. An interactive tool for comparing lists with Venn Diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html (2007).

Szklarczyk, D. et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 51, D638–d646 (2023).

pubmed: 36370105 doi: 10.1093/nar/gkac1000

Godfrey, J., Riscal, R., Skuli, N. & Simon, M. C. Glucagon signaling via supraphysiologic GCGR can reduce cell viability without stimulating gluconeogenic gene expression in liver cancer cells. Cancer Metab 10, 4 (2022).

pubmed: 35123542 pmcid: 8817478 doi: 10.1186/s40170-022-00280-1

Lim Kam Sian, T. C. C. et al. A Semiautomated Proteomics and Phosphoproteomics Protocol for the Identification of Novel Therapeutic Targets and Predictive Biomarkers in In Vivo Xenograft Models of Pediatric Cancers. Methods Mol. Biol. 2806, 229–242 (2024).

pubmed: 38676807 doi: 10.1007/978-1-0716-3858-3_17

Humphrey, S. J., Karayel, O., James, D. E. & Mann, M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat Protoc 13, 1897–1916 (2018).

pubmed: 30190555 doi: 10.1038/s41596-018-0014-9

Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11, 2301–2319 (2016).

pubmed: 27809316 doi: 10.1038/nprot.2016.136

Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 10, 1794–1805 (2011).

pubmed: 21254760 doi: 10.1021/pr101065j

Zhang, H. et al. Phospho-analyst: an interactive, easy-to-use web platform to analyze quantitative phosphoproteomics data. J Proteome Res. 22, 2890–2899 (2023).

pubmed: 37584946 doi: 10.1021/acs.jproteome.3c00186

Yu, K. et al. qPTM: an updated database for PTM dynamics in human, mouse, rat and yeast. Nucleic Acids Res. 51, D479–d487 (2023).

pubmed: 36165955 doi: 10.1093/nar/gkac820

Zaru, R. & Orchard, S. UniProt Tools: BLAST, Align, Peptide Search, and ID Mapping. Curr. Protoc 3, e697 (2023).

pubmed: 36943033 pmcid: 10034637 doi: 10.1002/cpz1.697

Kleiner, D. E. et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41, 1313–1321 (2005).

pubmed: 15915461 doi: 10.1002/hep.20701

Folch, J., Lees, M. & Stanley, S.G.H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol. Chem. 226, 497–509 (1957).

pubmed: 13428781 doi: 10.1016/S0021-9258(18)64849-5

Haas, D. et al. Differences of phenylalanine concentrations in dried blood spots and in plasma: erythrocytes as a neglected component for this observation. Metabolites 11, 680 (2021).

pubmed: 34677395 pmcid: 8537883 doi: 10.3390/metabo11100680