TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing

Overview

Journal J Proteome Res

Publisher American Chemical Society

Specialty Biochemistry

Date 2021 Apr 26

PMID 33900084

Citations 110

Authors

Jiaming Li

Zhenying Cai

Ryan D Bomgarden

Ian Pike

Karsten Kuhn

John C Rogers

Thomas M Roberts

Steven P Gygi

Joao A Paulo

Affiliations

Soon will be listed here.

Abstract

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

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References

Paulo J, Navarrete-Perea J, Thakurta S, Gygi S . TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms. J Proteome Res. 2018; 18(1):565-570. PMC: 6465125. DOI: 10.1021/acs.jproteome.8b00902. View

Huang D, Sherman B, Lempicki R . Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1):44-57. DOI: 10.1038/nprot.2008.211. View

Simpson L, Li J, Liaw D, Hennessy I, Oliner J, Christians F . PTEN expression causes feedback upregulation of insulin receptor substrate 2. Mol Cell Biol. 2001; 21(12):3947-58. PMC: 87057. DOI: 10.1128/MCB.21.12.3947-3958.2001. View

Meier F, Brunner A, Koch S, Koch H, Lubeck M, Krause M . Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer. Mol Cell Proteomics. 2018; 17(12):2534-2545. PMC: 6283298. DOI: 10.1074/mcp.TIR118.000900. View

Juric D, Castel P, Griffith M, Griffith O, Won H, Ellis H . Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor. Nature. 2014; 518(7538):240-4. PMC: 4326538. DOI: 10.1038/nature13948. View

Sethi I, Cai Z, Roberts T, Yuan G . Molecular Profiling Establishes Genetic Features Predictive of the Efficacy of the p110β Inhibitor KIN-193. Cancer Res. 2019; 79(17):4524-4531. PMC: 6726497. DOI: 10.1158/0008-5472.CAN-19-0588. View

Rauniyar N, Yates 3rd J . Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res. 2014; 13(12):5293-309. PMC: 4261935. DOI: 10.1021/pr500880b. View

Ogata K, Ishihama Y . Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC/MS/MS. Anal Chem. 2020; 92(12):8037-8040. DOI: 10.1021/acs.analchem.0c01695. View

Ogata T, Kozuka T, Kanda T . Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J Virol. 2003; 77(16):9000-7. PMC: 167236. DOI: 10.1128/jvi.77.16.9000-9007.2003. View

10.

Elias J, Gygi S . Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods. 2007; 4(3):207-14. DOI: 10.1038/nmeth1019. View

11.

Thorpe L, Yuzugullu H, Zhao J . PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer. 2014; 15(1):7-24. PMC: 4384662. DOI: 10.1038/nrc3860. View

12.

Bekker-Jensen D, Bernhardt O, Hogrebe A, Martinez-Val A, Verbeke L, Gandhi T . Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries. Nat Commun. 2020; 11(1):787. PMC: 7005859. DOI: 10.1038/s41467-020-14609-1. View

13.

Rad R, Li J, Mintseris J, OConnell J, Gygi S, Schweppe D . Improved Monoisotopic Mass Estimation for Deeper Proteome Coverage. J Proteome Res. 2020; 20(1):591-598. DOI: 10.1021/acs.jproteome.0c00563. View

14.

Schweppe D, Prasad S, Belford M, Navarrete-Perea J, Bailey D, Huguet R . Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal Chem. 2019; 91(6):4010-4016. PMC: 6993951. DOI: 10.1021/acs.analchem.8b05399. View

15.

Chang H, Cai Z, Roberts T . The Mechanisms Underlying PTEN Loss in Human Tumors Suggest Potential Therapeutic Opportunities. Biomolecules. 2019; 9(11). PMC: 6921025. DOI: 10.3390/biom9110713. View

16.

Humphrey S, Karayel O, James D, Mann M . High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform. Nat Protoc. 2018; 13(9):1897-1916. DOI: 10.1038/s41596-018-0014-9. View

17.

Savitski M, Wilhelm M, Hahne H, Kuster B, Bantscheff M . A Scalable Approach for Protein False Discovery Rate Estimation in Large Proteomic Data Sets. Mol Cell Proteomics. 2015; 14(9):2394-404. PMC: 4563723. DOI: 10.1074/mcp.M114.046995. View

18.

Navarrete-Perea J, Yu Q, Gygi S, Paulo J . Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3. J Proteome Res. 2018; 17(6):2226-2236. PMC: 5994137. DOI: 10.1021/acs.jproteome.8b00217. View

19.

Schweppe D, Rusin S, Gygi S, Paulo J . Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry. J Proteome Res. 2019; 19(1):554-560. PMC: 6996458. DOI: 10.1021/acs.jproteome.9b00759. View

20.

Li J, Van Vranken J, Vaites L, Schweppe D, Huttlin E, Etienne C . TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat Methods. 2020; 17(4):399-404. PMC: 7302421. DOI: 10.1038/s41592-020-0781-4. View