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PubMed2004

Characterization and molecular cloning of cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana.

Cellobiose dehydrogenase (CDH) was purified from the brown-rot fungus Coniophora puteana grown in culture containing crystalline cellulose as a carbon source. The purified enzyme gave a single band at 115 kDa on SDS-PAGE and showed a typical flavocytochrome absorption spectrum. The enzyme oxidized both cellobiose and cellooligosaccharides, but not their monomer, glucose, suggesting typical kinetic features of CDH. A cDNA encoding CDH was cloned by RT-PCR using primers designed from the consensus sequences of known CDHs from white-rot fungi. The cDNA consists of 2448 bp, including an open reading frame encoding the 18 amino acids of the putative signal peptide and the 756 amino acids of the mature protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data for tryptic fragments of the purified C. puteana CDH were consistent with partial amino acid sequences of the mature protein deduced from the cloned cDNA. Moreover, the sequences contained common characteristics of CDH, i.e., two possible residues for a heme ligand (Met 64 and His 160), a flavin-binding motif, and two glucose-methanol-choline oxidoreductase motifs. This is the first cloning of CDH from a brown-rot fungus, and the results suggest structural and kinetic similarity of C. puteana CDH to white-rot fungal CDHs.

Journal Article↗
PubMed2000

Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.

Journal Article↗
PubMed1994

Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli.

We have used thermodynamic and kinetic techniques to monitor the guanidinium chloride induced (GdmCl-induced) denaturation of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli (ePRAI). Although CD-monitored equilibrium denaturation curves are consistent with cooperative unfolding of the protein centered at 1.45 M GdmCl, fluorescence readings drop by over 25% in the region preceding the CD-monitored transition, suggesting non-two-state behavior. Kinetics experiments measure a slow relaxation rate with negative fluorescence amplitude when protein is diluted from 0 to 0.5 M GdmCl, corroborating results from equilibrium conditions. Detection of several unfolding and refolding rates in final GdmCl concentrations from 0 to 5.0 M indicates the presence of at least one intermediate along unfolding and refolding pathways. GdmCl dependence of the relaxation rates can be explained most easily by a nonsequential mechanism for ePRAI unfolding, though a sequential mechanism cannot be ruled out. The data corroborate the fragment complementation studies of Eder and Kirschner [Eder, J., & Kischner, K. (1992) Biochemistry 31, 3617-3625], which are consistent with unfolding of the C-terminal portion of a yeast-derived PRAI in its folding intermediate. In ePRAI, such partial unfolding would expose W391 to quenching by solvent molecules; W356, ePRAI's other tryptophan, is buried in the hydrophobic core and is unlikely to be affected by local changes in structure. A C-terminally unfolded folding intermediate has been demonstrated in the folding of tryptophan synthase (alpha-subunit), a related beta alpha-barrel enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗
PubMed2003

Humoral immune response to fibrillar beta-amyloid peptide.

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.

Alzheimer Disease↗
PubMed2003

Formation of aroma compounds from ribose and cysteine during the Maillard reaction.

The headspace volatiles produced from a phosphate-buffered solution (pH 5) of cysteine and a 1 + 1 mixture of ribose and [(13)C(5)]ribose, heated at 95 degrees C for 4 h, were examined by headspace SPME in combination with GC-MS. MS data indicated that fragmentation of ribose did not play a significant role in the formation of the sulfur aroma compounds 2-methyl-3-furanthiol, 2-furfurylthiol, and 3-mercapto-2-pentanone in which the carbon skeleton of ribose remained intact. The methylfuran moiety of 2-methyl-3-(methylthio)furan originated from ribose, whereas the methylthio carbon atoms came partly from ribose and partly from cysteine. In 3-mercapto-2-butanone one carbon unit was split from the ribose chain. On the other hand, all carbon atoms in 3-thiophenethiol stemmed from cysteine. In another trial cysteine, 4-hydroxy-5-methyl-3(2H)-furanone and [(13)C(5)]ribose were reacted under the same conditions. The resulting 2-methyl-3-furanthiol was mainly (13)C(5)-labeled, suggesting that it stems from ribose and that 4-hydroxy-5-methyl-3(2H)-furanone is unimportant as an intermediate. Whereas 2-mercapto-3-pentanone was found unlabeled and hence originated from 4-hydroxy-5-methyl-3(2H)-furanone, its isomer 3-mercapto-2-pentanone was formed from both 4-hydroxy-5-methyl-3(2H)-furanone and ribose. A new reaction pathway from ribose via its 1,4-dideoxyosone is proposed, which explains both the formation of 2-methyl-3-furanthiol without 4-hydroxy-5-methyl-3(2H)-furanone as an intermediate and a new way to form 3-mercapto-2-pentanone.

Carbon Isotopes↗
PubMed2005

An automated group contribution method in predicting aquatic toxicity: the diatomic fragment approach.

We developed a group contribution method (GCM) to correlate acute toxicity (96 h LC50) for the fathead minnow (Pimephales promelas) for 607 organic chemicals. Unlike most of the existing methods, the new one makes no use of predefined groups as descriptors. A simple general rule is proposed to break down any molecule into diatomic fragments. The entire data set was partitioned three times. Each time, a training set and a test set were obtained with a ratio of 2:1. For each partition quantitative structure-activity relationship, models were developed using Powell's minimization method, multilinear regression, neural networks, and partial least squares. The GCM method achieved a good correlation of the data for both training and test sets, regardless of the partition considered. The method is therefore robust and can be generally applied. Further model improvements are described.

Animals↗
PubMed2002

Detection of fowl adenovirus associated with hydropericardium hepatitis syndrome by a polymerase chain reaction.

Hydropericardium hepatitis syndrome (HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction (PCR) was developed to detect the fowl adenovirus (FAV) associated with HHS. The virus from infected livers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.

Animals↗
PubMed1997

Identification of parvalbumin alpha in bovine hypothalamus: a partial primary structure.

In the course of the study of structure-functional properties and molecular mechanisms of neuropeptides and of low molecular weight proteins of the central nervous system we succeeded in isolating from the soluble fraction of bovine hypothalamus a protein having M(r) 11897.3, according to mass spectral analysis. The purification procedure was mainly based on reversed phase HPLC. As the N-terminus of the molecule was found to be blocked, we have subjected it to CNBr degradation. By Edman microsequence analysis of the peptide fragments and by data base searching the isolated substance was identified as parvalbumin alpha (PRVA)-one of the calcium-binding proteins. However, its primary structure was found not to be identical to that of the known PRVAs from other sources. One of the features of PRVA is its stability. Being subjected to an exhausting purification procedure it retains its complete structure. As neuropeptides and low molecular weight proteins are found to be polyfunctional, a central question concerns the biological role of PRVAs in terms of "where and when" they express their action.

Amino Acid Sequence↗
PubMed1976

Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight.

Adipokinetic hormone, isolated from locust corpora cardiaca, has been identified as a blocked peptide: PCA-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2. The detailed structure is based on mass spectrometric data, substantiated in part by dansyl-Edman and carboxypeptidase data on thermolytic fragments. This is the first peptide hormone from an insect neuroendocrine organ to be fully characterised.

Amino Acid Sequence↗
PubMed2004

Oxidative-stress-related proteome changes in Helicobacter pylori-infected human gastric mucosa.

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. Proteomic analysis of the human gastric mucosa from the patients with erosive gastritis, peptic ulcer or gastric cancer, which were either infected or not with H. pylori, was used to determine the differentially expressed proteins by H. pylori in the human gastric mucosa in order to investigate the pathogenic mechanism of H. pylori -induced gastric diseases. Prior to the experiment, the expression of the main 18 proteins were identified in the gastric mucosa and used for a proteome map of the human gastric mucosa. Using two-dimensional electrophoresis of the protein isolated from the H. pylori -infected tissues, Coomassie Brilliant Blue staining and computerized analysis of the stained gel, the expression of eight proteins were altered in the H. pylori -infected tissues compared with the non-infected tissues. MS analysis (matrix-assisted laser desorption/ionization-time of flight MS) of the tryptic fragment and a data search allowed the the identification of the four increased proteins (78 kDa glucose-regulated protein precursor, endoplasmin precursor, aldehyde dehydrogenase 2 and L-lactate dehydrogenase B chain) and the four decreased proteins (intracellular chloride channel protein 1, glutathione S-transferase, heat-shock protein 60 and cytokeratin 8) caused by H. pylori infection in the gastric mucosa. These proteins are related to cell proliferation, carcinogenesis, cytoskeletal function and cellular defence mechanism. The common feature is that these proteins are related to oxidative-stress-mediated cell damage. In conclusion, the established gastric mucosal proteome map might be useful for detecting the disease-related protein changes. The H. pylori -induced alterations in protein expression demonstrate the involvement of oxidative stress in the pathogenesis of H. pylori -induced gastric diseases, including inflammation, ulceration and carcinogenesis.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1997

Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli.

The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.

Bacterial Proteins↗
PubMed1998

Vasoactive intestinal peptide shortens both G1 and S phases of neural cell cycle in whole postimplantation cultured mouse embryos.

Vasoactive intestinal peptide, a trophic and mitogenic factor, stimulates growth in whole cultured mouse embryos. Inhibition of this growth function between embryonic days 9 and 11 induces growth retardation accompanied by severe microcephaly. In the present study, to determine the effects of this peptide on the different phases of the cell cycle of neural cells, embryonic day 9.5 cultured mouse embryos were cumulatively labelled with bromodeoxyuridine. Vasoactive intestinal peptide (10(-7)M) shortened S phase and G1 phase of neuroepithelial cells by 50% (4.8-2.4 h) and 58% (1.9-0.8 h), respectively, compared with controls. G2 and M phases were not modified by vasoactive intestinal peptide treatment. Total cell cycle length was consequently reduced by 43% (8.2-4.7 h) in vasoactive intestinal peptide treated embryos, compared with controls. In contrast, vasoactive intestinal peptide did not modify the rate of neuroepithelial cell death as assessed by the proportion of nuclei containing fragmented DNA. These data suggest that vasoactive intestinal peptide stimulates growth in premigratory stages of nervous system development by shortening S and G1 phases of the cell cycle and that S phase duration can be regulated by a physiological peptide.

Animals↗
PubMed1979

Distinct H-2-linked Ir genes control both antibody and T cell responses to different determinants on the same antigen, myoglobin.

The murine antibody and T lymphocyte proliferative responses to sperm whale myoglobin (Mb) were found to be under control of two distinct H-2-linked immune response (Ir) genes (Ir-Mb-1, mapping in the I-A subregion, and Ir-Mb-2, mapping in I-C). H-2(d) mice (B10.D2 and DBA/2), with both genes, were high responders to Mb and its fragments for both antibody secretion and T cell proliferation, while H-2(b) (B10) and H-2(k) (B10.BR) mice were low responders. Strains with only Ir-Mb-2 [B10.A and B10.A(5R)], which were intermediate responders to Mb, made antibodies to and proliferated in response to the NH(2)-terminal fragment (1-55) but not the COOH-terminal fragment (132-153) when immunized with Mb. In contrast, mice carrying only the Ir-Mb-1 gene (D2.GD and B10.GD) made antibodies to and proliferated in response to both fragments. However, their proliferation to fragment (1-55) was often lower than that of their congenic high responders (DBA/2 and B10.D2, respectively), possibly because they respond to only some of the determinants on this NH(2)-terminal fragment. Thus, these data demonstrate that distinct Ir genes, mapping in separate I-subregions of H-2, control responses to different antigenic determinants on the same protein molecule. Moreover, the gene that controls the T lymphocyte responses to a given determinant also controls production of antibodies specific for that same determinant (or a closely associated one).

Animals↗
PubMed1981

Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.

Animals↗
PubMed1990

Sexual hybridization of Lycopersicon esculentum and Solanum rickii by means of a sesquidiploid bridging hybrid.

A sesquidiploid hybrid having two genomes of Lycopersicon esculentum and one of Solanum lycopersicoides served as a pistillate bridging parent in crosses with Solanum rickii to produce L. esculentum x S. rickii hybrid progeny. Of the four progeny obtained, one (GH2754) was diploid and three were aneuploid with extra S. lycopersicoides chromosomes. The hybrids had morphological features of both parents, but attributes of the wild parent dominated. The hybrid nature of the four progeny was confirmed by isozyme, restriction fragment length polymorphism, and cytological analyses. A mean of 9.15 bivalents was observed in pollen mother cells of GH2754. A high level of pollen abortion was seen in all hybrids. Crosses of the hybrids with staminate S. rickii yielded one backcross individual, revealing a very low, but certain level of female fertility. Colchicine treatment of GH2754 generated one promising amphidiploid hybrid, which exhibited strong preferential chromosome pairing (94% of the examined cells had 24 bivalents) and appreciable pollen fertility (43% stainable). Chromosome pairing, isozyme, and restriction fragment length polymorphism data support a very close relationship between the two Solanum spp. and a much greater distance between them and L. esculentum, but the data do not discriminate between them in respect to their distances from the latter. The cytological and molecular observations, previous reports of successful transfer of traits from S. lycopersicoides to L. esculentum, and our hybridization of L. esculentum x S. rickii suggest good prospects for gene transfer from S. rickii to L. esculentum.

Journal Article↗
PubMed1997

Thermodynamic analysis of the interaction between the 0.5beta Fv fragment and the RP135 peptide antigen derived from the V3 loop of HIV-1 gp120.

The Fv fragment of the 0.5beta monoclonal antibody has recently been constructed, expressed, and purified. It binds with nanomolar affinity to the immunogenic RP135 peptide that is derived from the principal neutralizing determinant of HIV-1 in the third hypervariable region of gp120. Here, we analyzed the temperature-dependence of binding of the 0.5beta Fv fragment to the RP135 peptide and a series of mutants thereof. Our results show that there is almost complete enthalpy-entropy compensation in the effects of mutations in the peptide on binding to the Fv, indicating that the mutations do not change the binding mechanism. There is good correlation, for residues within the antigenic epitope, between mutational effects on DeltaCp and calculated values of DeltaDeltaCp based on the extent of burial of polar and non-polar surface areas of amino acids. The value of DeltaCp for the binding of the 0.5beta Fv fragment to the wild-type RP135 peptide is found to be -5.0 (+/- 0.9) kcal K-1 mol-1 in the presence of 0.1% Tween-20 but only -0.1 (+/- 0.9) kcal K-1 mol-1 in its absence. This result has important implications for the successful application of the structural parameterization approach to predicting changes in heat capacity that accompany binding reactions carried out in the presence of detergent or protein-stabilizing agents.

Amino Acid Sequence↗
PubMed2004

Hypoxia-inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis.

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.

Apoptosis↗
PubMed2006

Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale.

We describe a novel LCMS approach to the relative quantitation and simultaneous identification of proteins within the complex milieu of unfractionated Escherichia coli. This label-free, LCMS acquisition method observes all detectable, eluting peptides and their corresponding fragment ions. Postacquisition data analysis methods extract both the chromatographic and the mass spectrometric information on the tryptic peptides to provide time-resolved, accurate mass measurements, which are subsequently used for quantitation and identification of constituent proteins. The response of E. coli to carbon source variation is well understood, and it is thus commonly used as a model biological system when validating an analytical method. Using this LCMS approach, we characterized proteins isolated from E. coli grown in glucose, lactose, and acetate. The change in relative abundance of the corresponding proteins was measured from peptides common to both conditions. Protein identities were also determined for those peptides that were unique to each condition, and these identities were found to be consistent with the underlying biochemical restrictions imposed by the growth conditions. The relative change in abundance of the characterized proteins ranged from 0.1- to 90-fold among the three binary comparisons. The overall coverage of the characterized proteins ranged from 10 to 80%, consisting of one to 34 peptides per protein. The quantitative results obtained from our study were comparable to other existing proteomic and transcriptional profiling approaches. This study illustrates the robustness of this novel LCMS approach for the simultaneous quantitative and comprehensive qualitative analysis of proteins in complex mixtures.

Chromatography, High Pressure Liquid↗