Characterization of the isoenzymes of pig-liver esterase. 1. Chemical Studies.

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis(4-nitro-[14C]phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals.     

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.     

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.     

稳定同位素化学标记结合质谱技术在定量蛋白质组学中的应用

传统的蛋白质组定量策略主要是通过双向凝胶电泳来进行相对定量。由于该方法不能对相对分子质量极高或极低、等电点极酸或极碱和含量低的蛋白质以及膜蛋白质等进行有效分离和检测,所以已不能适应目前蛋白质组研究深入发展的需要。近年来,定量蛋白质组学的发展主要是以同位素亲和标签试剂为代表的、以质谱检测为核心的稳定同位素化学标记方法。稳定同位素化学标记结合质谱技术,使定量蛋白质组的分析更趋简单、准确和快速,具有良好的发展前景。本文对稳定同位素化学标记结合质谱技术在定量蛋白质组学中的研究进展进行了评述。     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.     

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.     

18O labeling over a coffee break: a rapid strategy for quantitative proteomics

Proteomics-based quantification methods for differential protein expression measurements are among the most important and challenging techniques in the field of mass spectrometry. Though numerous quantification methods have been established, no method meets all the demands for measuring accurate protein expression levels. Of the various relative quantification methods by isotopic labeling, (18)O labeling method has been shown to be simple, specific, cost-effective and applicable to a wide range of analyses. However, some researchers refrain from using the method due to long incubation periods required during the labeling process. To address this problem, we demonstrate a method by which the labeling procedure can be completed in 15 min. We digested and labeled samples using immobilized trypsin on micro-spin columns to speed up the enzyme-mediated oxygen substitution, thereby completing the labeling process within 15 min with high labeling efficiency. We demonstrate the efficiency and accuracy of the method using a four protein mixture and whole cell lysate from rat vascular endothelial cells.     

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.     

Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.     

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS3 acquisition

Recently, we reported a novel proteomics quantitation scheme termed “combined precursor isotopic labeling and isobaric tagging (cPILOT)” that allows for the identification and quantitation of nitrated peptides in as many as 12–16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co‐isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple‐stage mass spectrometry (MS³) data acquisition, a selective MS³ method that focuses on product selection of the y₁ fragment of lysine‐terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS³ acquisition methods.     

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the α/β importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.     

Subunit structures of purified beef mitochondrial cytochromebc 1 complex from liver and heart

The existence of tissue-specific isozymes of cytochromec oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrialbc ₁ complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochromec oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels ofbc ₁ complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochromec oxidase,bc ₁ complexes from liver and heart do not exhibit tissue-specific differences.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation

Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.