Determination of the lipid composition of the GPI anchor.

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.

Introduction

Lipidation is an essential post-translational modification by which proteins are covalently modified with specific lipids that regulate protein localization, function, and stability. GPI anchoring is a special type of lipidation present in all eukaryotes that occurs at the endoplasmic reticulum (ER) and targets GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of essential physiological roles [1]. Immediately after GPI attachment and during GPI-AP secretory transport to the cell surface, the lipid moiety of the GPI anchor (GPI-lipid) undergoes structural remodeling, which is important for GPI-AP function and trafficking [2]. The initial structure and subsequent remodeling process of the precursor GPI-lipid moiety vary among proteins and species. In the yeast Saccharomyces cerevisiae, for instance, two different types of lipid moieties, diacylglycerol or ceramide, can be present in the GPI anchors on mature proteins [3].

We have used mass spectrometry to directly elucidate the structure of the GPI-lipid moiety of the specific GPI-AP, Gas1 [4]. Mass spectrometry-based methods have been extensively used for detailed analysis of posttranslational modifications. This methodology presents important advantages for GPI-lipid moiety analysis, such as higher precision and ease of use than classical techniques based on the incorporation of radioactive lipid precursors and subsequent detection of the proteins by fluorography [5]. Since identification by mass spectrometry of a single yeast GPI-AP, such as Gas1, requires prior purification of the protein, we need either a specific antibody or to use Gas1 tagged with GFP for immunopurification [6]. The latter makes the protocol applicable to a wider range of protein substrates. Yeast cells expressing Gas1-GFP are broken by glass beads and differentially centrifuged to generate a membrane fraction. Gas1-GFP is solubilized from the membrane fraction using the detergent digitonin and then affinity purified by the GFP-trap system. The immunopurified proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which contributes to completely remove any remnant of membrane lipids from the protein sample. Next, electrophoresed Gas1-GFP is transferred and immobilized on activated polyvinylidenedifluoride (PVDF) membrane by Western blotting. The PVDF membrane is treated with nitrous acid to deaminate the glucosamine residue of the GPI-glycan, a residue which occurs only rarely in mammalian glycans, but is part of the core structure, common to all GPIs. This chemical reaction is specific for the amino sugar and cleaves the protein and glycan attached to the GPI-glycan from the inositol-containing GPI-lipid [7], which remains attached to the PVDF membrane due to its hydrophobic nature. Finally, the GPI-lipid can be recovered by extraction with the negative mode solvent from the PVDF membrane for analysis by ESI-MS/MS mass spectrometry. For lipid species identification we use a multiple reaction monitoring (MRM) approach [8, 9].

The MRM is a targeted method used in tandem mass spectrometry. For each lipid molecular species, a parent ion and a product ion are defined. The parent ion is selected and fragmented to create a product ion that is finally detected. This is a highly selective and sensitive method that allows a rapid lipid profiling of the GPI-lipid. The disadvantage of the MRM is that requires a user-defined list of lipid species. Therefore, to decipher an uncharacterized lipid moiety a nontargeted profiling approach would be more suitable.

Materials and methods

“The protocol described in this peer-reviewed article is published on protocols.io and is included for printing as S1 File with this article.

Expected results

The protocol described here for analyzing the lipid composition of the GPI anchor was employed to address the role of the chain length of membrane ceramide in the sorting of GPI-APs upon exit from the endoplasmic reticulum (ER) in yeast [4]. In wild type cells, very long (C26) chain ceramide is present in the ER membrane and in the lipid moiety of the GPI anchor of the GPI-AP Gas1 [10]. We showed that newly synthesized Gas1-GFP is segregated from transmembrane secretory proteins and sorted into selective ER exit sites (ERES) during ER export. To tackle the relevance of membrane ceramide for ER sorting, we engineered a modified yeast strain (GhLag1) that produces cellular membranes with shorter ceramides (C16-C18) than in the wild-type strain (C26) [4, 11]. Mass spectrometry analysis revealed that although C18 and C16 ceramides are by far the major ceramides detected in GhLag1 membranes, the GPI anchor of Gas1-GFP expressed in GhLag1 strain contains C26 ceramide, the same lipid, as in wild-type [4]. Therefore, because in the GhLag1 strain the acyl chain length of membrane ceramides, but not the GPI ceramide is decreased, we could use this strain to specifically examine the role of the acyl chain length of membrane ceramides in ER sorting. We observed that Gas1-GFP expressed in GhLag1 failed to be sorted into selective ERES and, instead, was rerouted to exit the ER with transmembrane secretory proteins via common ERES. Based on these results, we concluded that ceramide acyl chain length in the ER membrane is an essential determinant for ER protein clustering and sorting [4].

We determined that in GhLag1 strain the GPI anchor of Gas1-GFP contains C26 ceramide using the method described here. For this purpose, Gas1-GFP was expressed under control of its own promoter in wild-type and GhLag1 cells and was then immunopurified from solubilized membranes with the GFP-trap system, applied to SDS-PAGE, transferred to a hydrophobic PVDF membrane and stained with amido black. The respective bands were cut and treated with nitrous acid to separate the inositolphosphoceramide (IPC) from the Gas1-GFP protein and the GPI-glycan. The separated IPC, that remains attached to the PVDF membrane due to its hydrophobicity, was extracted using the negative solvent for analysis by negative ion ES-MS/MS mass spectrometry. The sample was infused on a TSQ Vantage using a Triversa Nanomate. To provide a fast and accurate profile of the Gas1p lipid anchor we use an MRM approach applying a predefined list of known inositolphosphoceramide lipids of the yeast Saccharomyces cerevisiae (S1 Table). To obtain the published results, 5 biological replicates of blank, wild-type and Ghlag1 strains were done and the MRM list was read 3 times for each of them (technical replicates). As seen in Fig 1, the GPI-lipid of Gas1-GFP from wild-type and Ghlag1 strains specifically contains the lipid species IPC-B d/t44:0, an IPC with phytosphingosine (4-hydroxysphinganine) and a saturated C26 fatty acid. Therefore, our protocol provides qualitative data that shows that the GPI-lipid of Gas1 in the strain GhLag1 has a C26 ceramide. This method was developed for ceramide-type GPI-APs however, this could also be applied for a diacylglycerol-type phosphatidylinositol GPI-APs of yeast using the MRM list provided (S2 Table).

Fig 1

Mass spectrometry analysis of GPI lipid species of Gas1-GFP in wild-type and GhLag1 cells.

Isolated GPI lipids of Gas1-GFP from wild-type (B) and GhLag1 (C) cells and the blank sample (A) were detected by electrospray ionization and tandem mass spectrometry. The figure shows a representative cycle of transition measurements. The signal intensity of each lipid species was detected by multiple reaction monitoring. The GPI lipid from wild-type cells and Ghlag1 cells was identified as IPC with phytosphingosine and saturated C26 fatty acid (IPC-B d/t44:0) Time indicates the reading moment of each transition.

Step-by-step protocol, also available on protocols.io.

(PDF)

Click here for additional data file.

m/z of precursor/product ion for multiple-reaction monitoring of sphingolipid species of the yeast Saccharomyces cerevisiae used in this study.

(PDF)

Click here for additional data file.

m/z of precursor/product ion for multiple-reaction monitoring of phosphatidylinositol lipid species from the yeast Saccharomyces cerevisiae.

(PDF)

Click here for additional data file.

22 Jun 2021

PONE-D-21-17892

Determination of the lipid composition of the GPI anchor

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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.

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Reviewer #2: Yes: Roger J Morris

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21 Jul 2021

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.

Submitted filename: Aguilera-romero_response to reviewers.pdf

Click here for additional data file.

2 Aug 2021

Determination of the lipid composition of the GPI anchor

PONE-D-21-17892R1

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5 Aug 2021

PONE-D-21-17892R1

Determination of the lipid composition of the GPI anchor

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