Lozano-Prieto, MartaCamafeita, EmilioJorge, InmaculadaLaguillo-Gómez, AndreaBarrero-Rodríguez, RafaelDevesa, Cristina APertusa, ClaraCalvo, EnriqueSánchez-Madrid, FranciscoVázquez, JesúsMartin-Cofreces, Noa B2024-12-092024-12-092024-07-30J Proteomics. 2024 Jul 30:304:105229.https://hdl.handle.net/20.500.12105/25869This study was supported by the Spanish Ministry of Science and Innovation, Agencia Estatal de Investigación by competitive grants PID2021- 122348NB-I00, PID2022-141890B-I00, PID2020-120412RB-I00, PDC2021-121797-I00 and PGC2018-097019-BI00 funded by MICIU/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, PLEC2022-009298, PLEC2022-009235 and EQC2021-007053-P funded by MICIU/AEI/10.13039/501100011033 and by “European Union NextGenerationEU/ PRTR”, and S2022/BMD-7333-CM (INMUNOVAR-CM) and P2022/BMD7209 (INTEGRAMUNE) funded by Comunidad de Madrid. CIBER Cardiovascular (CB16/11/00272, CB16/11/00277) Fondo de Investigación Sanitaria del Instituto de Salud Carlos III; co-funding by Fondo Europeo de Desarrollo Regional (FEDER). The project leading to these results has received funding from ”La Caixa” Foundation under the project codes LCF/PR/HR22/52420019 and LCF/PR/HR23/52430018. MLP is supported by a FPI fellowship (PRE2021-097478). ALG is supported by a FPU fellowship (FPU18/03882). RBR is supported by a FPU fellowship (FPU20/03365). CAD is supported by a FPI fellowship (PRE2019-090019). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación Y Universidades (MICIU) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033).Mass-tolerant open search methods allow the high-throughput analysis of modified peptides by mass spectrometry. These techniques have paved the way to unbiased analysis of post-translational modifications in biological contexts, as well as of chemical modifications produced during the manipulation of protein samples. In this work, we have analyzed in-depth a wide variety of samples of different biological origin, including cells, extracellular vesicles, secretomes, centrosomes and tissue preparations, using Comet-ReCom, a recently improved version of the open search engine Comet-PTM. Our results demonstrate that glutamic acid residues undergo intensive methyl esterification when protein digestion is performed using in-gel techniques, but not using gel-free approaches. This effect was highly specific to Glu and was not found for other methylable residues such as Asp.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/Closed searchOpen searchPost-translational modificationProteomicsSystems biologyAttribution-NonCommercial-NoDerivatives 4.0 International38880355304Journal of Proteomicsopen access