Cardiac sarcoplasmic reticulum and sarcolemmal proteins separated by two-dimensional electrophoresis: surfactant effects on membrane solubilization.

We evaluated the use of the alkyaryl amidosulfobetaine zwitterionic detergent, designated as C8psi, to facilitate the solubilization of cardiac subcellular, membrane-associated proteins. Hearts from 7-week-old male Sprague-Dawley rats were isolated, and the left ventricles dissected and subsequently homogenized. The sarcolemma (SL) and the sarcoplasmic reticulum (SR) were isolated from different homogenate preparations originating from rat hearts by ultracentrifugation methods. The isolated membrane preparations were solubilized and the proteins precipitated. After resuspension, protein separation was achieved in first-dimensional IEF using an immobilized (pH 4-7) gradient and in the second dimension using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then stained, images analyzed, and protein spots excised for subsequent identification. Protein identification from both SR and SL samples did not identify any of the known major membrane-associated proteins. Solubilization of whole tissue lysates with C8psi resulted in no increase in the total number of proteins detected relative to samples solubilized in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulforate (CHAPS). The data suggest the utility of newer surfactants such as C8psi to improve both the resolution of (2-D) protein profiles and increase the number of proteins extracted from subcellular organelle fractions.

Application of mass spectrometry for target identification and characterization.

Mass spectrometry-based methodologies span the vast expanse of drug discovery. Both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) support proteomics-based research projects by identifying proteins separated and isolated by polyacrylamide gel electrophoresis. MALDI-MS-based surface scanning of one-dimensional isoelectric focusing gels, "virtual 2-D gel electrophoresis," represents a potentially high throughput means to map proteins and to determine protein profiles. Mass spectrometry can also be used to directly study the covalent and noncovalent interactions of drug molecules and biomolecule targets. Drug binding examples discussed include the binding of covalent and noncovalent inhibitors to src SH2 domain protein, and the interaction of aminoglycoside antibiotic neomycin and HIV Tat peptide-TAR RNA.

Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes.

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding.

Coupling capillary high-performance liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of peptides via automated microblotting onto membrane substrates.

To minimize low-quantity sample handling for protein sequencing and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a system consisting of an HPLC interfaced to an automated blotting device was used for off-line sample collection. Typically, protein digests are separated by reverse-phase HPLC and the resulting peptide fractions are pooled, concentrated, and then subjected to N-terminal sequence analysis. Obtaining unambiguous sequence from peptides derived from protein digestion at subpicomole levels requires careful sample handling to prevent loss of sample. In cases where multiple sequences are present, a secondary method such as mass spectrometry is needed to confirm the identity of the peptides. To minimize sample handling, commercial microblotting instruments have become available to deposit peptides directly onto polyvinylidene difluoride (PVDF) membrane for automated N-terminal sequence analysis. In order to adapt this technology to mass spectrometry, we investigated the use of MALDI-MS compatible membranes such as Teflon and polyethylene (PE) as the blotting media for fraction collection. Using a panel of standard peptides as well as protein digests, we demonstrate that peptides separated by capillary HPLC can be collected directly onto Teflon or PE and detected into the femtomole range. Furthermore, detailed sequence analysis could be obtained by postsource decay fragmentation spectra of individual peptides blotted onto either PE or Teflon. Due to the high sensitivity of the MALDI-MS from these membranes, it was discovered that the small amount of peptide that passed through the PVDF membrane during a collection of peptides for N-terminal sequencing was sufficient to be collected and mass analyzed from a second underlying MALDI-MS compatible membrane. Therefore, from a single HPLC separation, samples could be collected onto both PVDF for traditional N-terminal sequencing and PE or Teflon for MALDI-MS. We demonstrate the general utility of this method for sequencing peptides from a tryptic digestion at subpicomole levels and for identifying unknown proteins separated by 2-dimensional gel electrophoresis. The ability to generate both N-terminal sequence and confirmatory mass information from multiple peptides in a single separation greatly improves the reliability and the accuracy of protein characterization at subpicomole levels.

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Mass spectrometry of proteins directly from polyacrylamide gels.

The direct combination of thin-layer gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated with good sensitivity and mass accuracy, offering potential advantages in speed and reduced complexity. Mass spectra have been obtained from isoelectric focusing, sodium dodecyl sulfate, and native gels with as little as 660 fmol of α- and ß-chain bovine hemoglobin and 1 pmol of horse heart myoglobin loaded. CNBr digests were performed in situ, and the products were probed in-gel. Noncovalent complexes such as multimeric protein systems, enzyme inhibitor complexes, and protein-ligand complexes can also be characterized when gel electrophoresis is run under nondenaturing conditions. This approach shows promise for simplifying the interface between gel electrophoresis and mass spectrometry.