Mass Spectrometry-Based Analysis of Rat Liver and Hepatocellular Carcinoma Morris Hepatoma 7777 Plasma Membrane Proteome

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

Immunodetection of zona pellucida-like domain protein from salmon samples.

<p><b>A</b>, Coomassie stained SDS-polyacrylamide gel and B, corresponding Western blot of separated crude cupula extracts. Lane 1, untreated sample, lane 2, PNGase F treated sample with faster migration of zona pellucida-like domain protein. <b>C</b>, HE stained inner ear cross section, asterisk marks the cupula and the arrowhead the subcupulary region with sensory and supporting cells. <b>D</b>, immunostaining of inner ear cross section, the arrow probably indicates staining of supporting cells which produce the zona pellucida-like domain protein. This is even more pronounced in <b>E</b>.</p

Visualization of cupula proteins.

<p><b>A</b>, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. <b>B</b>, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the <i>N</i>-deglycosylation.</p

Peptide sequences obtained from the 45 kDa gel band from salmon.

<p>Data base searches were performed using Mascot and annotation of the MS/MS spectra was done manually. Amino-acid residues that differ from the published salmon sequence (C0H9B6) are bold. Amino-acid modifications: pyroQ, pyroglutamate, (delta mass: −17); ox, oxidized methionine (delta mass: +16); cam, carbamidomethyl, (delta mass: +57).</p>a<p>peptide with additional amino-acid exchange.</p><p>*peptide with additional modification.</p><p>Peptide sequences obtained from the 45 kDa gel band from salmon.</p

The cupula.

<p><b>A</b>, localization of the cupula in the inner ear. <b>B</b>, dissected cupula from salmon stained with Evans blue.</p