biostudies-literatureYe ZChinese Academy of Medical Sciences (CAMS)Novo Nordisk Fonden (Novo Nordisk Foundation)Novo NordiskVetenskapsrådet (Swedish Research Council)Innovationsfonden (Innovation Fund Denmark)499-509https://www.ebi.ac.uk/biostudies/studies/S-EPMC11903336biostudies-literatureUnknown22(3)Single-cell proteomics (SCP) promises to revolutionize biomedicine by providing an unparalleled view of the proteome in individual cells. Here, we present a high-sensitivity SCP workflow named Chip-Tip, identifying >5,000 proteins in individual HeLa cells. It also facilitated direct detection of post-translational modifications in single cells, making the need for specific post-translational modification-enrichment unnecessary. Our study demonstrates the feasibility of processing up to 120 label-free SCP samples per day. An optimized tissue dissociation buffer enabled effective single-cell disaggregation of drug-treated cancer cell spheroids, refining overall SCP analysis. Analyzing nondirected human-induced pluripotent stem cell differentiation, we consistently quantified stem cell markerNature methodsEnhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics.PMC119033362022-00323CELFFI-2022-0028432023-RC180-032022-I2M-2-004, 2023-I2M-2-005NNF14CC0001ERA-PerMed-JTC2022-OVA-PDMNNF22OC0076899NNF21OC0072070Seth AJoshi HJGuzman UHRodin SHorning OBvan der Hoeven LHuang HHuang MSabatier PBache NLechner MYYe ZPhlairaharn TLi XOlsen JVLiu ZHartlmayr DGrinnemo KHBekker-Jensen DBIzaguirre FfalseEnhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics.Single-cell proteomics (SCP) promises to revolutionize biomedicine by providing an unparalleled view of the proteome in individual cells. Here, we present a high-sensitivity SCP workflow named Chip-Tip, identifying >5,000 proteins in individual HeLa cells. It also facilitated direct detection of post-translational modifications in single cells, making the need for specific post-translational modification-enrichment unnecessary. Our study demonstrates the feasibility of processing up to 120 label-free SCP samples per day. An optimized tissue dissociation buffer enabled effective single-cell disaggregation of drug-treated cancer cell spheroids, refining overall SCP analysis. Analyzing nondirected human-induced pluripotent stem cell differentiation, we consistently quantified stem cell marker2025-01-01T00:00:00Z2025 Mar2025-04-03T23:56:53.035Z2025-04-03T23:56:53.035ZS-EPMC119033363982075010.1038/s41592-024-02558-2