{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T10:57:46Z","timestamp":1775559466232,"version":"3.50.1"},"reference-count":46,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2024,1,18]],"date-time":"2024-01-18T00:00:00Z","timestamp":1705536000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["RU 747\/1-2"],"award-info":[{"award-number":["RU 747\/1-2"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["TH1461\/7-2"],"award-info":[{"award-number":["TH1461\/7-2"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["FOR 2509"],"award-info":[{"award-number":["FOR 2509"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.<\/jats:p>","DOI":"10.3390\/ijms25021191","type":"journal-article","created":{"date-parts":[[2024,1,18]],"date-time":"2024-01-18T11:28:46Z","timestamp":1705577326000},"page":"1191","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9698-0773","authenticated-orcid":false,"given":"Roman","family":"Sakson","sequence":"first","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany"},{"name":"Heidelberg Biosciences International Graduate School (HBIGS), Heidelberg University, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lars","family":"Beedgen","sequence":"additional","affiliation":[{"name":"Center for Child and Adolescent Medicine, Department Pediatrics I, Heidelberg University, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1593-9769","authenticated-orcid":false,"given":"Patrick","family":"Bernhard","sequence":"additional","affiliation":[{"name":"Institute for Surgical Pathology, Medical Center\u2014University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9919-9838","authenticated-orcid":false,"given":"K. Merve","family":"Alp","sequence":"additional","affiliation":[{"name":"Max Delbr\u00fcck Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole","family":"L\u00fcbbehusen","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ralph","family":"R\u00f6th","sequence":"additional","affiliation":[{"name":"nCounter Core Facility, Institute of Human Genetics, University Hospital Heidelberg, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Beate","family":"Niesler","sequence":"additional","affiliation":[{"name":"nCounter Core Facility, Institute of Human Genetics, University Hospital Heidelberg, 69120 Heidelberg, Germany"},{"name":"Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcin","family":"Luzarowski","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1150-7344","authenticated-orcid":false,"given":"Olga","family":"Shevchuk","sequence":"additional","affiliation":[{"name":"Department of Immunodynamics, Institute of Experimental Immunology and Imaging, University Hospital Essen, 45147 Essen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthias P.","family":"Mayer","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Thiel","sequence":"additional","affiliation":[{"name":"Center for Child and Adolescent Medicine, Department Pediatrics I, Heidelberg University, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9152-2266","authenticated-orcid":false,"given":"Thomas","family":"Ruppert","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,1,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1111\/j.1749-6632.2010.05840.x","article-title":"Congenital disorders of glycosylation","volume":"1214","author":"Jaeken","year":"2010","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1038\/s41436-019-0647-2","article-title":"Therapeutic approaches in Congenital Disorders of Glycosylation (CDG) involving N-linked glycosylation: An update","volume":"22","author":"Verheijen","year":"2020","journal-title":"Genet. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1093\/glycob\/cwh072","article-title":"Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum","volume":"14","author":"Gao","year":"2004","journal-title":"Glycobiology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"65","DOI":"10.4052\/tigg.24.65","article-title":"Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis","volume":"24","author":"Takahashi","year":"2012","journal-title":"Trends Glycosci. Glycotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1016\/j.bbagen.2008.03.005","article-title":"Dolichol-phosphate mannose synthase: Structure, function and regulation","volume":"1780","author":"Maeda","year":"2008","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2430","DOI":"10.1016\/j.bbamcr.2013.04.001","article-title":"N-linked protein glycosylation in the ER","volume":"1833","author":"Aebi","year":"2013","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/j.molcel.2013.03.003","article-title":"elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats","volume":"50","author":"Buettner","year":"2013","journal-title":"Mol. Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1038\/nrm3383","article-title":"Vertebrate protein glycosylation: Diversity, synthesis and function","volume":"13","author":"Moremen","year":"2012","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Albuquerque-Wendt, A., Hutte, H.J., Buettner, F.F.R., Routier, F.H., and Bakker, H. (2019). Membrane Topological Model of Glycosyltransferases of the GT-C Superfamily. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20194842"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1038\/nmeth.2015","article-title":"Selected reaction monitoring-based proteomics: Workflows, potential, pitfalls and future directions","volume":"9","author":"Picotti","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1002\/mas.21540","article-title":"The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics","volume":"39","author":"Pino","year":"2020","journal-title":"Mass Spectrom. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1002\/pmic.201100463","article-title":"Using iRT, a normalized retention time for more targeted measurement of peptides","volume":"12","author":"Escher","year":"2012","journal-title":"Proteomics"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1021\/acs.jproteome.1c00749","article-title":"Skyline Batch: An Intuitive User Interface for Batch Processing with Skyline","volume":"21","author":"Marsh","year":"2022","journal-title":"J. Proteome Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1038\/nprot.2010.192","article-title":"Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics","volume":"6","author":"Geiger","year":"2011","journal-title":"Nat. Protoc."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"7042","DOI":"10.1016\/S0021-9258(19)39256-7","article-title":"The sequence and transcript heterogeneity of the yeast gene ALG1, an essential mannosyltransferase involved in N-glycosylation","volume":"265","author":"Albright","year":"1990","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"21828","DOI":"10.1074\/jbc.M010896200","article-title":"The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum","volume":"276","author":"Cipollo","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1093\/glycob\/3.4.357","article-title":"Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: The alg2 mutation","volume":"3","author":"Jackson","year":"1993","journal-title":"Glycobiology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1551","DOI":"10.1021\/ac403185a","article-title":"Combine and conquer: Surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins","volume":"86","author":"Waas","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"884","DOI":"10.1093\/bioinformatics\/btt607","article-title":"Protter: Interactive protein feature visualization and integration with experimental proteomic data","volume":"30","author":"Omasits","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/nprot.2009.57","article-title":"A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: Multiple reaction monitoring-initiated detection and sequencing (MIDAS)","volume":"4","author":"Unwin","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"100165","DOI":"10.1016\/j.mcpro.2021.100165","article-title":"An Introduction to Advanced Targeted Acquisition Methods","volume":"20","author":"Selbach","year":"2021","journal-title":"Mol. Cell. Proteom."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1074\/mcp.RA117.000543","article-title":"Panorama Public: A Public Repository for Quantitative Data Sets Processed in Skyline","volume":"17","author":"Sharma","year":"2018","journal-title":"Mol. Cell. Proteom."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4633","DOI":"10.1002\/pmic.200800326","article-title":"Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks","volume":"8","author":"Dupuis","year":"2008","journal-title":"Proteomics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2786","DOI":"10.1074\/jbc.RA118.005642","article-title":"The intramembrane protease SPP impacts morphology of the endoplasmic reticulum by triggering degradation of morphogenic proteins","volume":"294","author":"Avci","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1016\/j.tig.2018.03.002","article-title":"Perspectives on Glycosylation and Its Congenital Disorders","volume":"34","author":"Ng","year":"2018","journal-title":"Trends Genet."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"22498","DOI":"10.1074\/jbc.M302850200","article-title":"A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis","volume":"278","author":"Thiel","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1002\/humu.22019","article-title":"Improved diagnostics lead to identification of three new patients with congenital disorder of glycosylation-Ip","volume":"33","author":"Thiel","year":"2012","journal-title":"Hum. Mutat."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2596","DOI":"10.1091\/mbc.E15-03-0168","article-title":"Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly","volume":"26","author":"Mueller","year":"2015","journal-title":"Mol. Biol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1007\/s00216-012-6203-4","article-title":"Quantitative mass spectrometry in proteomics: Critical review update from 2007 to the present","volume":"404","author":"Bantscheff","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1016\/j.ejmg.2017.10.012","article-title":"Congenital disorders of glycosylation (CDG): Quo vadis?","volume":"61","author":"Peanne","year":"2018","journal-title":"Eur. J. Med. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1093\/brain\/awt010","article-title":"Congenital myasthenic syndromes due to mutations in ALG2 and ALG14","volume":"136","author":"Cossins","year":"2013","journal-title":"Brain"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1016\/j.nmd.2013.12.010","article-title":"Clinical and pathological heterogeneity of a congenital disorder of glycosylation manifesting as a myasthenic\/myopathic syndrome","volume":"24","author":"Monies","year":"2014","journal-title":"Neuromuscul. Disord."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s10719-021-09976-w","article-title":"Mass spectrometry glycophenotype characterization of ALG2-CDG in Argentinean patients with a new genetic variant in homozygosis","volume":"38","author":"Papazoglu","year":"2021","journal-title":"Glycoconj. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1074\/mcp.M200025-MCP200","article-title":"Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics","volume":"1","author":"Ong","year":"2002","journal-title":"Mol. Cell. Proteom."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"966","DOI":"10.1093\/bioinformatics\/btq054","article-title":"Skyline: An open source document editor for creating and analyzing targeted proteomics experiments","volume":"26","author":"MacLean","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"10116","DOI":"10.1021\/ac102179j","article-title":"Effect of collision energy optimization on the measurement of peptides by selected reaction monitoring (SRM) mass spectrometry","volume":"82","author":"MacLean","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/0003-2697(84)90782-6","article-title":"A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids","volume":"138","author":"Wessel","year":"1984","journal-title":"Anal. Biochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1896","DOI":"10.1038\/nprot.2007.261","article-title":"Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips","volume":"2","author":"Rappsilber","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1074\/mcp.TIR118.000953","article-title":"FlashPack: Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS","volume":"18","author":"Kovalchuk","year":"2019","journal-title":"Mol. Cell. Proteom."},{"key":"ref_40","unstructured":"R Development Core Team (2018). R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e16950","DOI":"10.7554\/eLife.16950","article-title":"Global, quantitative and dynamic mapping of protein subcellular localization","volume":"5","author":"Itzhak","year":"2016","journal-title":"eLife"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc. Ser. B"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2524","DOI":"10.1093\/bioinformatics\/btu305","article-title":"MSstats: An R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments","volume":"30","author":"Choi","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_44","unstructured":"UniProt Consortium (2022). UniProt: The Universal Protein Knowledgebase in 2023. Nucleic Acids Res., 51, D523\u2013D531."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1016\/j.cell.2016.06.041","article-title":"Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome","volume":"166","author":"Kusebauch","year":"2016","journal-title":"Cell"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"M110.003384","DOI":"10.1074\/mcp.M110.003384","article-title":"CONSeQuence: Prediction of reference peptides for absolute quantitative proteomics using consensus machine learning approaches","volume":"10","author":"Eyers","year":"2011","journal-title":"Mol. Cell. Proteom."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/2\/1191\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:49:47Z","timestamp":1760104187000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/2\/1191"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,18]]},"references-count":46,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["ijms25021191"],"URL":"https:\/\/doi.org\/10.3390\/ijms25021191","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.09.15.291732","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,1,18]]}}}