Abstract
Isobaric peptide termini labeling (IPTL) is an approach for quantitative proteomics based on crosswise isotopic labeling of peptides at the N- and C-terminus. The labeling reagents are chosen in isotopic variations that the resulting mass of all labels per peptide is isobaric, but the individual label on each peptide terminus is different. Therefore, the quantitative difference of the peptide signal can be determined by the fragment ions of the corresponding MS2 spectra. Here, we describe an approach for triplex-IPTL to allow the comparison of three proteomes. This approach is based on digestion of the proteins by endoproteinase Lys-C, followed by three combinations of selective dimethylation of the peptide N-termini and subsequent dimethylation of the lysine residues at the C-termini. Data analysis is performed using Mascot for database searches and the freely available software package IsobariQ for quantification.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ankney JA, Muneer A, Chen X (2018) Relative and absolute quantitation in mass spectrometry-based proteomics. Annu Rev Anal Chem 11:40–77
Koehler CJ, Strozynski M, Kozielski F et al (2009) Isobaric peptide termini labeling for MS/MS-based quantitative proteomics. J Proteome Res 8:4333–4341
Waldbauer J, Zhang L, Rizzo A, Muratore D (2017) diDO-IPTL: a peptide labeling strategy for precision quantitative proteomics. Anal Chem 89:11498–11504
Cao T, Zhang L, Zhang Y et al (2017) Site-specific quantification of protein ubiquitination on MS2 fragment ion level via isobaric peptide labeling. Anal Chem 89:11468–11475
Koehler CJ, Arnzten MØ, de Souza GA, Thiede B (2013) An approach for triplex-isobaric peptide termini labeling (Triplex-IPTL). Anal Chem 85:2478–2485
Arntzen MØ, Koehler CJ, Barsnes H, Berven FS et al (2011) IsobariQ: software for isobaric quantitative proteomics using IPTL, iTRAQ, and TMT. J Proteome Res 10:913–920
Koehler CJ, Arnzten MØ, Treumann A, Thiede B (2012) Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL). Bioanal Anal Chem 404:1103–1114
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Koehler, C.J., Thiede, B. (2021). An Approach for Triplex-IPTL. In: Marcus, K., Eisenacher, M., Sitek, B. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 2228. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1024-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1024-4_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1023-7
Online ISBN: 978-1-0716-1024-4
eBook Packages: Springer Protocols