Mass spectrometry-based analysis of rat liver and hepatocellular carcinoma Morris hepatoma 7777 plasma membrane proteome

Abstract

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues.

Figure 1. Experimental scheme of the high-throughput analyses of isolated plasma membranes of rat liver and hepatocellular carcinoma Morris hepatoma 7777

Isolated plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were pretreated with either 1) Salt (sodium carbonate) washing only or 2) sodium carbonate washing followed by trypsin predigestion to remove the soluble and peripheral membrane proteins. Subsequently, the resulting plasma membrane pellets were solubilized with detergents. Proteins were reduced, alkylated, and then directly incorporated into a polyacrylamide gel. The enhanced gel-assisted digestion was then performed. Peptides extracted from the gel were applied to LC-MS/MS analysis for protein identification and quantification. 3) Deglycosylation was performed after the formation of tube-gel to increase the protein sequence coverage for glycoprotein identification.

Figure 2. Representation of the distributions of proteins or protein groups with predicted TM helices identified from normal rat liver plasma membranes (LIPM) or hepatocellular carcinoma Morris hepatoma 7777 plasma membranes (MHPM) using different treatments

Isolated plasma membranes were 1) LIPM Control: LIPM directly analyzed using the nonelectrophoretic gel-assisted digestion approach, 2) LISW or MHSW: LIPM or MHPM were pretreated with carbonate washing, 3) LIPD or MHPD: LIPM or MHPM treated with carbonate washing followed by predigestion with trypsin. After the enhanced gel-assisted digestion, tryptic peptides were subjected to LC-MS/MS analysis for protein identification and quantification. Both data acquired by a QSTAR XL mass spectrometer (left) and an Orbitrap Velos mass spectometer (right) are shown for comparison. The putative transmembrane helices were predicted using the transmembrane hidden Markov model (TMHMM) algorithm for each identified protein. Distributions of proteins (above) or protein groups (below) with their predicted TM helices are both presented.

Figure 3. Histogram illustrations of the distributions of proteins with predicted TM helices identified from normal rat liver and hepatocellular carcinoma Morris hepatoma plasma membrane using different treatments

Both data acquired by a QSTAR XL mass spectrometer (left) and an Orbitrap Velos mass spectometer (right) are shown for comparison. Identified proteins predicted with more than three TM helices are also shown in a more detailed view (below). Isolated plasma membranes were 1) LIPM Control: normal rat liver plasma membrane directly analyzed using the nonelectrophoretic gel-assisted digestion approach, 2) LISW or MHSW: LIPM or MHPM treated with carbonate washing, 3) LIPD or MHPD: LIPM or MHPM treated with carbonate washing followed by predigestion with trypsin. After the enhanced gel-assisted digestion, tryptic peptides were analyzed by LC-MS/MS for protein identification and quantification.

Figure 4. Comparisons of proteins predicted with one or more TM helix identified from normal rat liver and hepatocellular carcinoma Morris hepatoma plasma membrane using different treatments

The data shown here were acquired on an Orbitrap Velos mass spectrometer. Venn diagram representations (left) and histogram representations (right) of identified proteins predicted with one or more TM helix were represented for a) b) LISW and LIPD, and c) d) MHSW and MHPD.