Dataset Information

Deuterium-free, Three-plexed Peptide Di-Ethylation for Highly Accurate Quantitative Proteomics

ABSTRACT: The deuterium, frequently used stable isotope in isotopic labeling for quantitative proteomics, could deteriorate the accuracy and precision of proteome quantification owing to the retention time shift of deuterated peptides from the hydrogenated counterpart. Herein, we introduce a novel three-plexed peptide ‘di-ethylation’ using only 13C-isotopologues of acetaldehyde and demonstrate that the accuracy and precision of our method in proteome quantification are significantly superior to the conventional deuterium-based di-methylation labeling in both a single shot and multidimensional LC-MS/MS analysis of HeLa proteome. Furthermore, in time-resolved profiling of Xenopus laevis early embryogenesis, our 3-plexed di-ethylation outperformed isobaric labeling approaches in terms of quantification accuracy or number of protein identification, generating >2 times more differentially expressed proteins. Our cost-effective and highly accurate 3-plexed di-ethylation method could contribute to various types of quantitative proteomics applications, in which three of multiplexity would be sufficient.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Jae Hun Jung

LAB HEAD: Jaehun Jung

PROVIDER: PXD012117 | Pride | 2019-01-17

REPOSITORIES: Pride

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Publications

Deuterium-Free, Three-Plexed Peptide Diethylation for Highly Accurate Quantitative Proteomics.

Jung Jaehun J Jeong Kyowon K Choi Yeon Y Kim Sun Ah SA Kim Hyunjoon H Lee Joon Won JW Kim V Narry VN Kim Kwang Pyo KP Kim Jong-Seo JS

Journal of proteome research 20190129 3

The deuterium, a frequently used stable isotope in isotopic labeling for quantitative proteomics, could deteriorate the accuracy and precision of proteome quantification owing to the retention time shift of deuterated peptides from the hydrogenated counterpart. We introduce a novel three-plexed peptide "diethylation" using only <sup>13</sup>C isotopologues of acetaldehyde and demonstrate that the accuracy and precision of our method in proteome quantification are significantly superior to the co ...[more]

PMID: 30638020

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Project description:The epithelial growth factor receptor (EGFR) signalling network plays an essential role inproliferation and survival of colorectal cancer cells. For this reason, EGFR is targeted using therapeutic antibodies blocking EGFR signalling (e.g. cetuximab). However, anti-EGFR therapy is rarely curative as subpopulations of tumor cells survive the initial treatment and develop therapy-resistance. To analyse cellular signalling and assess the quantitative changes of the proteome and the phosphoproteome several proteomics approaches have been established. Here, we compared the widely used quantification strategies label-free (LF), SILAC and TMT and systematically evaluated their technical characteristics including coverage, technical variability, complementarity and the ability to assess statistical significant differences. We studied the dynamics of the EGFR signalling network upon treatment withcetuximab after 0 h, 3 h and 24 h, representing the first cellular adaption towards anti-EGFR therapy, with the aim to identify the most powerful approach for monitoring cellular signalling in this setup. We demonstrate that LF had a superior coverage regarding quantification of both, the proteome and phosphoproteome, while SILAC exhibited the highest precision resulting in superior assessment of differentially abundant proteins and class I phosphosites (p-sites). TMT was outperformed by the other two approaches, illustrating its critical dependency on the applied labelling-design. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated after 3 h and 24 h. The dynamics of significantly regulated p-sites illustrated an initial downregulation of the EGFR and MAPK signalling pathways, which was partially rescued as soon as after 24h. We identified upregulation and signalling via ERBB3 as a possible mechanism bypassing the blockage of EGFR. Moreover, phosphorylation motifs associated to calcium signalling were continuously upregulated, thereby representing compensatory signalling presumably resulting in the observed reactivation of MAPK1/3 (Erk1/2).

Dataset Information

Deuterium-free, Three-plexed Peptide Di-Ethylation for Highly Accurate Quantitative Proteomics

Publications

Deuterium-Free, Three-Plexed Peptide Diethylation for Highly Accurate Quantitative Proteomics.

Similar Datasets

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