Abstract
BackgroundThe low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps.ResultsWe have devised a simple protocol using a 'tube-gel' protein digestion that, when combined with mass spectrometry, can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain. Lipid rafts (detergent-resistant membranes using Triton X-100 extraction) prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation. After in-gel digestion of proteins, nanospray LC-MS/MS was used to analyze the extracted peptides, and the resulting spectra were searched to identify the proteins present in the sample. Using the standard 'label-free' proteomics approach, the total number of MS/MS spectra for the identified proteins was used to provide a measure of relative protein abundances. This approach was successfully applied to lipid rafts prepared from neonatal mouse brain. A total of 216 proteins were identified: 127 proteins (58.8%) were predicted to be membrane proteins, or membrane-associated proteins and 175 proteins (~80%) showed less than a 2-fold variation in the relative abundance in replicate samples.ConclusionThe tube-gel protein digestion protocol coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) offers a simple and reproducible method for identifying and quantifying the changes of relative abundances in lipid raft proteins from neonatal mouse brain and could become a useful approach for studying lipid raft proteins from various tissues.
Highlights
The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins
The lipid raft fractions 2~4 were highly enriched in sterols, SM, and Cer compared to plasma membranes
We have successfully combined a 'tube-gel' protein digestion protocol with nanospray LC-Mass spectrometry (MS)/MS analysis to carry out a high throughput proteomic mapping of lipid raft proteins isolated from neonatal mouse brain
Summary
The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. Lipid raft samples prepared by different methods are composed of highly enriched lipids and low concentrations of hydrophobic proteins. Raft preparations contain many non-proteinaceous substances including exogenous reagents, such as salts, buffers and detergents employed for sample preparation These highly enriched lipids and non-protein components, or contaminants can often interfere with proteome analysis and their removal is a critical step before any proteome analysis can be performed. The low protein concentrations in raft samples do not present a limitation for analysis, methods used for removing lipids and other interfering substances from raft samples can lead to excessive protein loss. The process of lipid raft preparation suitable for mass spectrometry is a major factor in the variability of data obtained by these powerful proteomic techniques
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