Determination of the lipid composition of the GPI anchor

Auxiliadora Aguilera-Romero; Susana Sabido-Bozo; Sergio Lopez; Alejandro Cortes-Gomez; Sofia Rodriguez-Gallardo; Ana Maria Perez-Linero; Isabelle Riezman; Howard Riezman; Manuel Muñiz

doi:10.1371/journal.pone.0256184

Peer Review History

Original SubmissionMay 30, 2021
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Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The protocol article written by Aguilera-Romero et al described the lipid analysis of GPI-anchored proteins. The authors showed an analytical method of inositol phosphoceramide structures on GFP-tagged Gas1 proteins expressed in Saccharomyces cerevisiae. It would be useful for readers who are interested in lipid species of GPI-anchors and their changes by mutations in genes. The reviewer recommends the publication after the appropriate revision. 1) In the abstract, the authors wrote “an improved protocol”. Please describe more clearly what is improved compared to the previous methods (Fontaine et al. (2003) Glycobiology 13(3):169-77; Yoko-o et al. (2013) Mol. Microbiol. 88(1):140-55). Compared to the old methods, purification of PI using silica column was omitted. Is it because of changing the detection method using MRM in the analysis by ESI-MS/MS? 2) The authors listed the ions for MRM of lipids in Table 1. In the list, not only IPC species, but also MIPC and M(IP)2C species are listed. MIPC and M(IP)2C should be removed from the list, since these lipid species are not observed in the GPI lipids. Instead, is it possible to add diacylglycerol-type PI species? Reviewer #2: This protocol, presented here at the detail of a laboratory protocol, provides a highly standard series of methods to identify the lipid attached to an immunoaffinity tagged GPI-anchored protein. The force of these methods to identify lipid attachments to GPI-proteins is shown fully in their recent publication in Science Advances 2020;6(49). Developed in yeast, such is the importance of identifying lipid modification in all species that I am confident this protocol will be used widely in the future. Reviewer #3: The authors provide an excellent job of summarizing the significance and relevance of improving the protocol for detection of GPI-AP in yeast. This includes a detailed explanation of the roles of GPI and the distinctions between that of yeast and mammalian. Additionally, a comprehensive description of the rational for use of mass spectrometry-based methods versus the use of traditional methods. The authors' observations regarding the significance of using the ceramide acyl chain length for the ER protein. There are a few clarifications that I am looking for: 1) How many samples were used for each the wild type and GhLag1, and just one blank were used? Were any biological or technical replicated used? 2) Which instrument/columns were used for MS/MS? Thank you. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Roger J Morris Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0256184.r001
Revision 1
August 2, 2021 Decision Letter - Michael Polymenis, Editor Determination of the lipid composition of the GPI anchor PONE-D-21-17892R1 Dear Dr. Muñiz, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Michael Polymenis, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0256184.r003
July 21, 2021 Author Response Point by Point response Reviewer #1: The protocol article written by Aguilera-Romero et al described the lipid analysis of GPI-anchored proteins. The authors showed an analytical method of inositol phosphoceramide structures on GFP-tagged Gas1 proteins expressed in Saccharomyces cerevisiae. It would be useful for readers who are interested in lipid species of GPI-anchors and their changes by mutations in genes. The reviewer recommends the publication after the appropriate revision. We thank the reviewer for the very positive and helpful comments on our manuscript, and we hope to have answered all concerns raised. 1) In the abstract, the authors wrote “an improved protocol”. Please describe more clearly what is improved compared to the previous methods (Fontaine et al. (2003) Glycobiology 13(3):169-77; Yoko-o et al. (2013) Mol. Microbiol. 88(1):140-55). Compared to the old methods, purification of PI using silica column was omitted. Is it because of changing the detection method using MRM in the analysis by ESI-MS/MS? Response: As pointed out by the reviewer, the adjective “improved” is not appropriate because our protocol is a compilation of methods used in Fontaine et al. (2003) Glycobiology 13(3):169-77, Yoko-o et al. (2013) Mol. Microbiol. 88(1):140-55 and Mehlert et al. (2009) Glycoconj J. 26(8):915-921. Therefore, we remove this adjective from the abstract. Following the method used by Mehlert et al. (2009) we decided to directly infuse the sample on the MS/MS without a previous purification by silica column. Thanks to the use of a targeted approach, MRM, we could clearly identify the lipid specie of the GPI anchor despite of background noise. This simplifies the method allowing more routine studies. However, for a more explorative approach we consider the protocol described in Yoko-o et al. (2013) to be more suitable. The text is now corrected as suggested by the reviewer. 2) The authors listed the ions for MRM of lipids in Table 1. In the list, not only IPC species, but also MIPC and M(IP)2C species are listed. MIPC and M(IP)2C should be removed from the list since these lipid species are not observed in the GPI lipids. Instead, is it possible to add diacylglycerol-type PI species? Response: In Rodriguez-Gallardo et al. (2020) (Sci Adv 6(50):eaba8237) we studied the role of very long (C26) chain membrane ceramides in protein sorting from the ER. For this purpose, we used a modified yeast strain GhLag1, that produces cellular membranes with only long (C18) rather than very long (C26) chain ceramides (Epstein et al. (2012) Mol Microbiol 84, 1018). Gas1, used in this work as a reporter, is a GPI-AP that has a C26 ceramide lipid in its GPI-anchor (Yoko-o et al. (2013) Mol. Microbiol. 88(1):140-55). Since the strain GhLag1 mainly produces C18 ceramides we wanted to determine whether Gas1 incorporates a C18 or a C26 ceramide in its GPI anchor. To achieve this, we developed the present method and applied a MRM list used routinely in our lab to define the sphingolipid composition of yeast strains, which contains IPC, MIPC and MIP2C lipid species. We agree with the reviewer comment that MIPC and MIP2C species are not required for our purpose, but we want to show the exact method used in the published paper that corresponds with the figure provided. Additionally, we think that the suggestion provided by the reviewer could be of interest to determine the lipid of the GPI-anchor therefore we provide an additional S2 table that contains a diacylglycerol-type PI MRM list. Reviewer #2: This protocol, presented here at the detail of a laboratory protocol, provides a highly standard series of methods to identify the lipid attached to an immunoaffinity tagged GPI-anchored protein. The force of these methods to identify lipid attachments to GPI-proteins is shown fully in their recent publication in Science Advances 2020;6(49). Developed in yeast, such is the importance of identifying lipid modification in all species that I am confident this protocol will be used widely in the future. We thank the reviewer for the appreciation of our manuscript. Reviewer #3: The authors provide an excellent job of summarizing the significance and relevance of improving the protocol for detection of GPI-AP in yeast. This includes a detailed explanation of the roles of GPI and the distinctions between that of yeast and mammalian. Additionally, a comprehensive description of the rational for use of mass spectrometry-based methods versus the use of traditional methods. The authors' observations regarding the significance of using the ceramide acyl chain length for the ER protein. There are a few clarifications that I am looking for: We thank the reviewer for the useful comments, and we hope to have answered the questions with the following. 1) How many samples were used for each the wild type and GhLag1, and just one blank were used? Were any biological or technical replicated used? Response: To obtain the results published in Rodriguez-Gallardo et al. (2020) (Sci Adv 6(50):eaba8237) we performed five experiments, each of them with its blank, Wild-type and Ghlag1 sample. Thus, we obtained a total of 5 biological replicates by sample. For each of them each transition of the MRM list was read three times (three technical replicates). We have added this information to the manuscript. 2) Which instrument/columns were used for MS/MS? The mass spectrometer used in our study is a TSQ Vantage and samples were infused on it using a Triversa Nanomate. We have added this information to the manuscript. Attachments Attachment Submitted filename: Aguilera-romero_response to reviewers.pdf https://doi.org/10.1371/journal.pone.0256184.r002
Formally Accepted
August 5, 2021 Acceptance Letter - Michael Polymenis, Editor PONE-D-21-17892R1 Determination of the lipid composition of the GPI anchor Dear Dr. Muñiz: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Michael Polymenis Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0256184.r004

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