A detergent‐ and cyanogen bromide‐free method for integral membrane proteomics: Application to Halobacterium purple membranes and the human epidermal membrane proteome

Abstract

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model

“…These two sets of peptides were identified as apolipoprotein A1, which has recently been reported as a biomarker for the detection of early stage ovarian cancer [21], and ␣-1-antitrypsin 1-1, which has been observed to have increased levels in lung cancer patients [22], demonstrating the facile ability of the present methodology to provide both quantitation and identification of putative biomarkers in the same high-throughput experiment. We hypothesize that efficient 18 O incorporation, as indicated by the mass spectra in Figure 5, may be attributed to the observed increase (ϳ20%) in trypsin activity in 20% (vol/vol) CH 3 OH as previously demonstrated by a N-benzoyl-L-arginine ethyl ester trypsin activity assay [23].…”

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

“…[16][17][18] The use of MS-compatible organic solvents such as methanol for membrane protein solubilization, followed by direct insolution tryptic digestion and LC-MS/MS, proved effective in proteomic studies of bacterial and human membranes. 17,[19][20][21][22][23] The application of this protocol to plant membrane proteomics has not been reported, although differential solubilization of membrane proteins in chloroform/methanol mixtures has been widely used in the extraction of highly hydrophobic proteins of the chloroplast envelope and mitochondrial membrane. 3,7,24 This procedure is biased toward hydrophobic membrane proteins and is often combined with extraction by basic and saline solutions to increase the diversity of membrane proteins obtained.…”

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

“…In addition to the interference encountered with mass spectrometric detection, concentrated urea or detergents denature many proteases and decrease their activity to cleave proteins. There are a number of reports describing technical advances to enable membrane protein analysis using the shotgun proteomics approach in which proteins are dissolved in a buffer containing surfactants or in an organic solvent, followed by protein digestion and liquid chromatography (LC) tandem mass spectrometry (MS/MS) analysis of the peptides [3][4][5][6][7][8][9][10][11][12]. Protein digestion was done with trypsin [3][4][5][6][7], cyanogen bromide (CNBr)-mediated enzymatic digestion [8 -11], and nonspecific proteinase K [12].…”