Limits...
Flagellar membranes are rich in raft-forming phospholipids.

Serricchio M, Schmid AW, Steinmann ME, Sigel E, Rauch M, Julkowska D, Bonnefoy S, Fort C, Bastin P, Bütikofer P - Biol Open (2015)

Bottom Line: Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells.Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella.Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry & Molecular Medicine, University of Bern, Bern 3012, Switzerland peter.buetikofer@ibmm.unibe.ch mauro.serricchio@utoronto.ca.

No MeSH data available.


Identification of phospholipid classes by MS. Representative total ion spectra of the most intense species of PC (A), PE (B), PI (C), PS (D), PG (E) and IPC (F). Parent ion spectra in positive (PC) or negative (PE, PI, PS, PG and IPC) ionisation mode are shown in the left panel, and the corresponding MS/MS fragment spectra are depicted in the right panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582118&req=5

BIO011957F3: Identification of phospholipid classes by MS. Representative total ion spectra of the most intense species of PC (A), PE (B), PI (C), PS (D), PG (E) and IPC (F). Parent ion spectra in positive (PC) or negative (PE, PI, PS, PG and IPC) ionisation mode are shown in the left panel, and the corresponding MS/MS fragment spectra are depicted in the right panel.

Mentions: The lipid composition of whole cells (containing all membranes, e.g. plasma membrane, endoplasmic reticulum, Golgi, nucleus, mitochondrion, glycosomes, acidocalcisomes) and purified flagella after tetracycline-induced down-regulation of Tb927.10.2880 was determined in two completely independent experiments. In each experiment, lipids were extracted and analysed by reverse-phase liquid chromatography high-resolution tandem mass spectrometry (LC-MS/MS) using a LTQ Orbitrap in both negative and positive ionisation modes. The separation of phospholipids by liquid chromatography was highly reproducible and allowed simultaneous acquisition of fragmentation spectra to facilitate identification of molecular species. Masses were acquired with a resolution of 60,000 with less than 1 ppm error, allowing identification of phospholipids based on their exact masses. Fig. 2A and B depict representative total ion chromatograms of lipid extracts of flagella (top panel) and whole cells (bottom panel) in negative (Fig. 2A) and positive (Fig. 2B) ionisation modes. Averaged total ion spectra across the retention time range 10–45 min from chromatograms in Fig. 2A and B are represented in Fig. 2C and D, respectively. Peak lists were extracted and subjected to several database searches (see Materials and Methods). Identification of individual phospholipid molecular species was based on collision-induced (CID) fragmentation using characteristic fragmentation patterns for the different phospholipid classes (Fig. 3). Using this LC-MS/MS protocol, we were able to identify a total of 216 phospholipid species in the negative and 193 species in the positive ionisation mode, with the majority of phospholipid classes and molecular species (>50%) confirmed by CID fragmentation. Two independent experiments were conducted, and only phospholipids identified in both experiments were considered real hits and used in our analyses. The resolution power of the liquid chromatography protocol is demonstrated in Fig. 4, showing the elution profiles of the major 36-carbon PC species in positive mode (panel A) and the major 36-carbon ether-type PE species in negative mode (panel B). The liquid chromatography method separated isobaric phospholipids with different types of linkages, allowing identification of alkyl-acyl versus alk-enyl-acyl (plasmalogen) molecular species (see Fig. 4B). We will first describe the complete set of lipids identified and then compare their relative abundances between whole cells and purified flagella.Fig. 2.


Flagellar membranes are rich in raft-forming phospholipids.

Serricchio M, Schmid AW, Steinmann ME, Sigel E, Rauch M, Julkowska D, Bonnefoy S, Fort C, Bastin P, Bütikofer P - Biol Open (2015)

Identification of phospholipid classes by MS. Representative total ion spectra of the most intense species of PC (A), PE (B), PI (C), PS (D), PG (E) and IPC (F). Parent ion spectra in positive (PC) or negative (PE, PI, PS, PG and IPC) ionisation mode are shown in the left panel, and the corresponding MS/MS fragment spectra are depicted in the right panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582118&req=5

BIO011957F3: Identification of phospholipid classes by MS. Representative total ion spectra of the most intense species of PC (A), PE (B), PI (C), PS (D), PG (E) and IPC (F). Parent ion spectra in positive (PC) or negative (PE, PI, PS, PG and IPC) ionisation mode are shown in the left panel, and the corresponding MS/MS fragment spectra are depicted in the right panel.
Mentions: The lipid composition of whole cells (containing all membranes, e.g. plasma membrane, endoplasmic reticulum, Golgi, nucleus, mitochondrion, glycosomes, acidocalcisomes) and purified flagella after tetracycline-induced down-regulation of Tb927.10.2880 was determined in two completely independent experiments. In each experiment, lipids were extracted and analysed by reverse-phase liquid chromatography high-resolution tandem mass spectrometry (LC-MS/MS) using a LTQ Orbitrap in both negative and positive ionisation modes. The separation of phospholipids by liquid chromatography was highly reproducible and allowed simultaneous acquisition of fragmentation spectra to facilitate identification of molecular species. Masses were acquired with a resolution of 60,000 with less than 1 ppm error, allowing identification of phospholipids based on their exact masses. Fig. 2A and B depict representative total ion chromatograms of lipid extracts of flagella (top panel) and whole cells (bottom panel) in negative (Fig. 2A) and positive (Fig. 2B) ionisation modes. Averaged total ion spectra across the retention time range 10–45 min from chromatograms in Fig. 2A and B are represented in Fig. 2C and D, respectively. Peak lists were extracted and subjected to several database searches (see Materials and Methods). Identification of individual phospholipid molecular species was based on collision-induced (CID) fragmentation using characteristic fragmentation patterns for the different phospholipid classes (Fig. 3). Using this LC-MS/MS protocol, we were able to identify a total of 216 phospholipid species in the negative and 193 species in the positive ionisation mode, with the majority of phospholipid classes and molecular species (>50%) confirmed by CID fragmentation. Two independent experiments were conducted, and only phospholipids identified in both experiments were considered real hits and used in our analyses. The resolution power of the liquid chromatography protocol is demonstrated in Fig. 4, showing the elution profiles of the major 36-carbon PC species in positive mode (panel A) and the major 36-carbon ether-type PE species in negative mode (panel B). The liquid chromatography method separated isobaric phospholipids with different types of linkages, allowing identification of alkyl-acyl versus alk-enyl-acyl (plasmalogen) molecular species (see Fig. 4B). We will first describe the complete set of lipids identified and then compare their relative abundances between whole cells and purified flagella.Fig. 2.

Bottom Line: Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells.Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella.Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry & Molecular Medicine, University of Bern, Bern 3012, Switzerland peter.buetikofer@ibmm.unibe.ch mauro.serricchio@utoronto.ca.

No MeSH data available.