Abstract
Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ong S-E, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
Qu Z, Meng F, Bomgarden RD et al (2014) Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents. J Proteome Res 13:3200–3211
Vajrychova M, Salovska B, Pimkova K et al (2019) Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology. Redox Biol 24:101227
Roumeliotis TI, Weisser H, Choudhary JS (2019) Evaluation of a dual isolation width acquisition method for isobaric labeling ratio decompression. J Proteome Res 18:1433–1440
Rauniyar N, Yates JR (2014) Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res 13:5293–5309
Shakir S, Vinh J, Chiappetta G (2017) Quantitative analysis of the cysteine redoxome by iodoacetyl tandem mass tags. Anal Bioanal Chem 409:3821–3830
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Tyanova S, Temu T, Sinitcyn P et al (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740
Lössner C, Warnken U, Pscherer A et al (2011) Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions. Anal Biochem 412:123–125
Acknowledgments
This work was supported by the Grant Agency of the Czech Republic, Project No. 15-03379S, Institutional Grant (Project No. RVO 68378050), and MH CZ-DRO (UHHK, 00179906).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Vajrychova, M., Salovska, B., Pimkova, K., Fabrik, I., Hodny, Z. (2023). SILAC-IodoTMT for Assessment of the Cellular Proteome and Its Redox Status. In: Luque-Garcia, J.L. (eds) SILAC. Methods in Molecular Biology, vol 2603. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2863-8_21
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2863-8_21
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2862-1
Online ISBN: 978-1-0716-2863-8
eBook Packages: Springer Protocols