Improved Recovery and Identification of Membrane Proteins from Rat Hepatic Cells using a Centrifugal Proteomic Reactor

Abstract

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with >2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

“…28 We then use DTT and iodoacetamide for protein reduction and alkylation, activate the trypsin by increasing pH to 8, and elute the resulting peptides in alkaline buffer for LC–MS/MS analysis. 28 Using this gel-free methodology, we have been able to detect 945 plasma membrane proteins and 955 microsomal membrane proteins in hepatic cells. 28 More than 800 out of these 945 and 955 proteins were not identified by the conventional in-gel digestion approach.…”

“…28 Using this gel-free methodology, we have been able to detect 945 plasma membrane proteins and 955 microsomal membrane proteins in hepatic cells. 28 More than 800 out of these 945 and 955 proteins were not identified by the conventional in-gel digestion approach. 28 We anticipate that these improvements will further expand upon this study’s identification of a mitochondrial protein-connexin interactome.…”

“…28 More than 800 out of these 945 and 955 proteins were not identified by the conventional in-gel digestion approach. 28 We anticipate that these improvements will further expand upon this study’s identification of a mitochondrial protein-connexin interactome. Application will require further optimization as we have found that the phospholipid-Cx32 interactions 29 in our fractionated microdomains complicate the required pH-dependent binding to cationic beads.…”

Proteomics Analysis of Human Obesity Reveals the Epigenetic Factor HDAC4 as a Potential Target for Obesity

“…Extracts were centrifuged at 14,000 rpm for 30 minutes at 4°C. Protein concentration was determined by Bradford method using γ-globulin as a standard and proteins were subjected to centrifugal proteomic reactor as described previously [23]. Briefly, 20 µg of proteins were added to 10 µL of strong cationic exchange (BcMag™ SCX Magnetic bead slurry (Bioclone, San Diego, CA) in 1 ml of 5% formic acid and the mixture was vigorously vortexed.…”

Bioanalysis of Eukaryotic Organelles

“…Zhou et al simplified and employed a previously developed proteomic reactor 260 to characterize the ER and Golgi microsomal membrane proteomes of rat hepatic cells (McA-RH7777). 261 The reactor itself consists of a 1.5-mL Eppendorf tube in which strong cation exchange beads are added to a lysed, acidified, and delipidated membrane pellet. It is then able to preconcentrate, derivatize, and support enzymatic digestion of the proteins.…”