High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip.
Animals
Biomarkers
/ analysis
Cell Line
Equipment Design
Lab-On-A-Chip Devices
Mice
Nanostructures
/ chemistry
Proteins
/ analysis
Proteomics
/ instrumentation
RAW 264.7 Cells
Reproducibility of Results
Sequence Analysis, RNA
Single-Cell Analysis
/ instrumentation
Specimen Handling
/ instrumentation
Tandem Mass Spectrometry
/ methods
Workflow
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
29 10 2021
29 10 2021
Historique:
received:
23
04
2021
accepted:
12
10
2021
entrez:
30
10
2021
pubmed:
31
10
2021
medline:
1
12
2021
Statut:
epublish
Résumé
Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.
Identifiants
pubmed: 34716329
doi: 10.1038/s41467-021-26514-2
pii: 10.1038/s41467-021-26514-2
pmc: PMC8556371
doi:
Substances chimiques
Biomarkers
0
Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
6246Subventions
Organisme : NIGMS NIH HHS
ID : P41 GM103493
Pays : United States
Organisme : NHLBI NIH HHS
ID : U01 HL122703
Pays : United States
Organisme : NHLBI NIH HHS
ID : U01 HL148860
Pays : United States
Organisme : NCI NIH HHS
ID : U24 CA210955
Pays : United States
Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
Références
Bioinformatics. 2012 Mar 15;28(6):882-3
pubmed: 22257669
Anal Chem. 2020 Feb 4;92(3):2665-2671
pubmed: 31913019
Sci Rep. 2020 Mar 12;10(1):4570
pubmed: 32165681
Mol Cell Proteomics. 2020 Nov;19(11):1739-1748
pubmed: 32847821
FEBS Lett. 2005 Oct 24;579(25):5631-4
pubmed: 16213494
Cell. 2016 Jul 28;166(3):755-765
pubmed: 27372738
Anal Chem. 2018 Apr 17;90(8):5430-5438
pubmed: 29551058
Methods Mol Biol. 2021;2185:159-179
pubmed: 33165848
Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7738-43
pubmed: 18509061
Nat Methods. 2017 May;14(5):513-520
pubmed: 28394336
Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13688-93
pubmed: 21808013
Nat Rev Mol Cell Biol. 2019 May;20(5):285-302
pubmed: 30659282
Science. 2015 Jan 23;347(6220):1260419
pubmed: 25613900
Cell Syst. 2017 Apr 26;4(4):458-469.e5
pubmed: 28396000
Mol Cell Proteomics. 2020 May;19(5):828-838
pubmed: 32127492
Cell Rep. 2018 Nov 20;25(8):2044-2052.e5
pubmed: 30463003
J Biol Chem. 2005 Mar 25;280(12):11665-74
pubmed: 15677455
Mol Syst Biol. 2022 Mar;18(3):e10798
pubmed: 35226415
Genome Biol. 2018 Oct 22;19(1):161
pubmed: 30343672
Nat Commun. 2018 Feb 28;9(1):882
pubmed: 29491378
Compr Physiol. 2012 Jan;2(1):1-29
pubmed: 23728969
Anal Chem. 2018 May 1;90(9):5810-5817
pubmed: 29648445
Anal Chem. 2020 Aug 4;92(15):10588-10596
pubmed: 32639140
Sci Rep. 2016 May 09;6:25533
pubmed: 27156886
Genome Biol. 2021 Jan 27;22(1):50
pubmed: 33504367
Elife. 2019 Nov 04;8:
pubmed: 31682227
Nat Protoc. 2016 Dec;11(12):2301-2319
pubmed: 27809316
Nat Methods. 2021 Jan;18(1):76-83
pubmed: 33288958
Nat Commun. 2019 Mar 6;10(1):1076
pubmed: 30842418
Lab Invest. 2017 Jan;97(1):4-13
pubmed: 27869795
Anal Chem. 2017 Jan 3;89(1):822-829
pubmed: 27959506
Chem Sci. 2020 Nov 17;12(3):1001-1006
pubmed: 34163866
Anal Chem. 2021 Jan 26;93(3):1658-1666
pubmed: 33352054
Mol Cell Proteomics. 2019 Oct;18(10):1967-1980
pubmed: 31332098
Nat Rev Chem. 2020 Mar;4(3):143-158
pubmed: 37128021
Nat Commun. 2021 Jun 7;12(1):3341
pubmed: 34099695
Anal Chem. 2020 Apr 7;92(7):4711-4715
pubmed: 32208662
Cell Rep. 2016 Jan 12;14(2):380-9
pubmed: 26748716
Nat Methods. 2016 Sep;13(9):731-40
pubmed: 27348712
Anal Chem. 2018 Dec 4;90(23):14003-14010
pubmed: 30375851
Nature. 2016 Sep 14;537(7620):347-55
pubmed: 27629641
ACS Nano. 2016 Nov 22;10(11):10173-10185
pubmed: 27788331
Am J Respir Crit Care Med. 2019 Feb 15;199(4):496-507
pubmed: 30290132
Anal Chem. 2019 Oct 15;91(20):13119-13127
pubmed: 31509397
Cell Mol Life Sci. 2013 Oct;70(19):3695-708
pubmed: 23619613
Angew Chem Int Ed Engl. 2018 Sep 17;57(38):12370-12374
pubmed: 29797682