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Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10(7) down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti(4+), our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research."],"pubmed_title":["Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography."],"pubmed_authors":["Loroch Stefan S,Zahedi René Peiman RP,Sickmann Albert A,","Loroch Stefan S, Zahedi René Peiman RP, Sickmann Albert A"],"name_synonyms":["solid, Chromatographies., sensitive"],"description_synonyms":["data, scale tissue, Bhlha41, determination, peltate hair, AUTSX5, DXS648, bHLHa48, backward, CCN3, QM, SCXB, SCXA, solid, Experiment, resilient, IGFBP-9, sensitive, PIG7, tough, BB114693, 3.1.3.48, NOV, scales, PlexA1, strong, Plxn1, scale, Step, plant peltate hair, PTPSTEP, chemical analysis., Striatum-enriched protein-tyrosine phosphatase, IGFBP9, mKIAA4053, IBP-9, Neural-specific protein-tyrosine phosphatase, L10, STEP, C130088N23Rik, 2600013D04Rik, assay, DXS648E, NOVh, Chromatographies, SIMPLE, Scl, reversed, TP53I7"],"pubmed_title_synonyms":["solid, Chromatographies., sensitive"],"pubmed_abstract_synonyms":["scale tissue, wide/broad, Activity, Bhlha41, Procedures, determination, H-PAST, Laboratory, Effects, peltate hair, ug, Longterm Effect, Phosphorylations, bHLHa48, Research Priorities, broad, backward, Procedure, mg, SCXB, phosphorylation, Cell, Long Term, SCXA, inadequate, polypeptide, period, solid, Polypeptides, Priority, resilient, sensitive, Method, PIG7, tough, BB114693, chemical analysis, Long Term Effects, Research Activities, 3.1.3.48, Studies, techniques, PAST, Research Activity, scales, Laboratory Research, Effect, sensitivity, Research and Development, Priorities, strong, scale, Research, Step, Longterm, cell, plant peltate hair, PTPSTEP, HeLa, Specificity, µg, Striatum-enriched protein-tyrosine phosphatase, proteins, Methodological, procedures, Long-Term, PAST1, Methodological Study, Research Activities., sample population, Longterm Effects, Activities, Study, wide, Neural-specific protein-tyrosine phosphatase, Methodological Studies, Specificity and Sensitivity, STEP, sample, Sensitivity, HPAST1, RCB0007, Long-Term Effect, assay, Development and Research, Research Priority, Chromatographies, SIMPLE, Long-Term Effects, time, Scl, reversed, methodology, TP53I7"],"view_count":["24"],"citation_count":["0"],"search_count":["5"],"full_dataset_link":["http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM32320005256"],"search_domains":["dbgap_ncbi~0","patentfamilies~0","rfam~0","merops~0","complex-portal~0","uniprot~0","wormbaseparasite~0","embl-covid19~0","reactome~0","emdb~0","wgs_masters~0","ebiweb_resources~0","opentargets_genetics~0","biomodels_all~0","ipd-mhc~0","ebiweb_teams~0","taxonomy~0","genome_assembly~0","sc-experiments~0","ebiweb_people~0","enzymeportal_enzymes~0","ipd-nhkir~0","cellosaurus~0","pdbe~0","chebi~0","patentproteins~0","interpro7~0","uniref~0","chembl~0","pdbekb~0","gpcrdb~0","hgnc~0","sc-genes~0","intact~0","rhea~0","ebiweb_training~0","alphafold~0","imgt-hla~0","patentnucleotides~0","ensemblroot~0","eva_studies~0","non-coding~0","europepmc~0","pubmed~1","identifiers_registry~0","pdbechem~0","hpa-covid19~0","eva-variants-covid19~0","biosamples~0","gwas_catalog~0","biotools~0","tls_masters~0","mesh~0","coding~0","sra~0","opentargets~0","efo~0","embl-pathogen~0","project~0","pride~1","human_diseases~0","geo_datasets~0","embl~0","treefam~0","uniparc~0","ols~0","dgva~0","intenz~0","go~0","tsa_masters~0","biosamples-covid19~0","ebiweb_corporate~0","omim~0","lrg~0","earlycause-molecular-sequences~0","ipd-kir~0","empiar~0","rnacentral~0","orcid_data_claims~0","gpmdb~2","lineage-covid19~0","metagenomics~0","pfam~0","pride archive~1","varsite~0"],"reanalysis_count":["0"],"submitter_keywords":["Resource Reanalysis"],"citation_count_scaled":["0.0"],"reanalysis_count_scaled":["0.0"],"view_count_scaled":["0.007402837754472548"],"download_count_scaled":["0.0"],"normalized_connections":["1.0"],"additional_accession":[]},"is_claimable":false,"name":"Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography.","description":"Data from ProteomeXchange, PXD ID: PXD001381. Experiment: ERLIC2, file: Velos02_06349 ERLIC2 F14.mzml. Published as part of Anal Chem. 2014 Nov 18  . From the Abstract: {{i}} ... Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for -g-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid phase-extraction step by strong cation exchange (SCX) or reversed phase (RP) and LC-MS analysis ... {{/i}}","dates":{"submission":"2014-12-09"},"accession":"GPM32320005256","cross_references":{"pubmed":["25405705"],"Pride":["PXD001381"],"pride":[],"Pride Archive":["PXD001381"]}}