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. 2017 May 10;7:1713. doi: 10.1038/s41598-017-01974-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic illustration of the strategy used for comparative phosphoproteomic analysis of D. melanogaster adults that were cold acclimated (CA) versus control (CO). For each replicate (n = 4), lysate was collected from 20 virgin females and 4 mg of proteins were subjected to proteolytic digestion. Tryptic peptides were desalted and enriched for phosphopeptides using three sequential steps: IMAC with an acidic elution (fraction 1), IMAC with basic elution (fraction 2), and TiO₂ on the IMAC flow through (fraction 3). Three different MS methods were then used for each elution fraction: a classical Collision-Induced Dissociation (CID), a Neutral Loss (NL) and a Multi-Stage Activation (MSA).