Strategies employing non‐gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT‐MS) are gaining attention as alternatives to ...two‐dimensional gel electrophoresis (2‐DE). We have conducted a large‐scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT‐MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT‐MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion‐trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under‐represented small proteins (<10 kDa) and failed to show clear superiority over 2‐DE methods in monitoring hydrophobic proteins from cell lysates.
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 β-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.
ABSTRACT
Depressed sarcoplasmic reticulum (SR) Ca‐cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or ...decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca‐cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics‐based analysis of PLN knockout (PLN‐KO) hearts in comparison to isogenic wild‐types. By use of 2‐dimensional gel electrophoresis (2‐DE), ˜3300 distinct protein spots were detected in either wild‐type or PLN‐KO ventricles. Protein spots observed to be altered between PLN‐KO and wild‐type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI‐TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two‐dimensional 32Pautoradiography was performed to analyze the phosphorylation profiles of PLN‐KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN‐KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN‐KO mouse in the long term.
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological ...inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)−N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.
Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the ...proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins.
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.
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.
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