A Proteomic Approach to Screening of Dynamic Changes in Detergent-Resistant Membranes from Activated Human Primary T Cells

Moltu, Kristine; Bjørgo, Elisa; Solstad, Therese; Berge, Torunn; Thiede, Bernd; Taskén, Kjetil

doi:10.4172/jpb.1000264

“…The use of more than one method to extract proteins from the lipid raft fractions may yield a more complete recovery of proteins [25]. Both MetI and MetII have been used extensively in other studies characterizing lipid raft proteomes from different tissues and cell types [1,3,12,14,73,74]. In the present study, extraction of proteins from the lipid raft fractions using MetI or MetII resulted in the identification of proteins common to both methods, as well as proteins unique to each method.…”

Section: Discussionmentioning

confidence: 99%

“…The strategy applied in MetI eliminates the sucrose from the enriched lipid raft fractions prior to MS analysis [16]. The procedure used in MetII mixes the sample with another detergent (OGP), followed by methanol-chloroform extraction, which aids in removing the interfering lipids that could be aggregated with the isolated proteins [73,74]. Extraction using MetII yielded a higher number of total proteins, as well as those with lipid modifications typical of membrane raft proteins [1,47].…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells

Filho

¹

,

Jaca

²

,

Baeza

³

et al. 2019

IJMS

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Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

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“…The use of more than one method to extract proteins from the lipid raft fractions may yield a more complete recovery of proteins [25]. Both MetI and MetII have been used extensively in other studies characterizing lipid raft proteomes from different tissues and cell types [1,3,12,14,73,74]. In the present study, extraction of proteins from the lipid raft fractions using MetI or MetII resulted in the identification of proteins common to both methods, as well as proteins unique to each method.…”

Section: Discussionmentioning

confidence: 99%

“…The strategy applied in MetI eliminates the sucrose from the enriched lipid raft fractions prior to MS analysis [16]. The procedure used in MetII mixes the sample with another detergent (OGP), followed by methanol-chloroform extraction, which aids in removing the interfering lipids that could be aggregated with the isolated proteins [73,74]. Extraction using MetII yielded a higher number of total proteins, as well as those with lipid modifications typical of membrane raft proteins [1,47].…”

Section: Discussionmentioning

confidence: 99%

“…In the analysis of the GO class cellular component, the proteins associated with terms other than plasma membrane may be explained in part by the fact that many proteins originally located in the cytoplasm or nucleus could be translocated and interact with plasma membrane constituents to form protein complexes [81]. Moreover, lipid rafts may also be found in cellular compartments other than the plasma membrane such as in endoplasmic reticulum and mitochondria [55,74,[82][83][84]. Several lipids and proteins associated with lipid rafts are synthesized in the endoplasmic reticulum/Golgi apparatus before being transported to the plasma membrane [27], where they may move laterally within the plasma membrane as well as traffic continuously between the plasma membrane and internal compartments [78].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Lipid raft associated molecules modulate signal transduction and inflammatory mediator release in mast cells

Filho¹,

Garcia²

0

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À minha estimada orientadora, Profa. Dra. Maria Célia Jamur, pela atenção, pelos ensinamentos, pelas discussões e sugestões tão importantes para a realização deste trabalho. Muito obrigado pela oportunidade de crescimento e formação científica, pela confiança, pelo suporte e principalmente toda consideração. A senhora é exemplar e muito especial para mim. À minha segunda orientadora, Profa. Dra. Constance Oliver, meus sinceros agradecimentos pela sua participação no trabalho e também na minha formação, pelas dicas valiosas, pelo tempo dedicado às leituras e correções, e também pelos seus abraços tão especiais de apoio e carinho.

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