Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

Abstract

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure--beginning with isolated membrane fraction and finishing with MS data acquisition--takes 4-5 d.

SASH1 Is a Scaffold Molecule in Endothelial TLR4 Signaling

“…Jirik, University of Calgary, Calgary, AB, Canada). A rabbit polyclonal Ab to human SASH1 was raised against a synthetic peptide conjugated to keyhole limpet hemocyanin to recognize amino acids [8][9][10][11][12][13][14][15][16][17][18][19][20] at the N terminus of the deduced SASH1 protein sequence (University of British Columbia, Vancouver, BC, Canada). The SASH1 Ab was purified by protein A-Sepharose chromatography.…”

“…Peptide pools were combined and lyophilized prior to strong cation exchange fractionation. Strong cation exchange fractionation was performed using an HP 1090 LC system (Agilent Technologies, Santa Clara, CA) as previously described (16). Sixteen fractions were collected, lyophilized, and reconstituted in 0.1% trifluoroacetic acid prior to nanoflow reversed-phase liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry (MS) (LTQ-FT; Thermo Fisher Scientific, Waltham, MA), as previously described (17).…”

Lipid-induced Muscle Insulin Resistance Is Mediated by GGPPS via Modulation of the RhoA/Rho Kinase Signaling Pathway

“…After a thorough wash, the samples were eventually subjected to immunoblotting analysis using the antiprenyl antibody (Abcam). To determine the membrane association of RhoA, subcellular fractionation was performed using ultracentrifugation as previously described (14). In brief, the cell pellets or tissues were lysed and homogenized with ice-cold Dounce tissue homogenizer, and the lysates were centrifuged at 100,000 ϫ g for 30 min (4°C).…”

Pressure Cycling Technology-assisted Protein Digestion for Efficient Proteomic Analysis

“…9 The use of mixedsolvent buffers containing various concentrations of organic solvents (e.g., methanol, acetonitrile, isopropanol) resulted in protein denaturing as well as enhanced solubility of hydrophobic proteins. 10 More recently, alternative energy inputs such as microwave energy 11 or high-intensity focused ultrasound 12 have been applied to digestions to further increase enzyme reaction rates. These approaches reduced the time required for digestion to several minutes using microwave-assisted digestion and to 15 -30 s using high-intensity focused ultrasound.…”

“…They were useful for solution 13 or in gel 14 digestions and were shown to be applicable to complex protein mixtures. 10 High pressure is another alternative energy that has been used to increase enzymatic activity. 15 High pressure is thought to change the protein conformation and force the penetration of water molecules into the protein interior, especially into cavities, leading to denaturing of the protein.…”