Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients.

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent "top-down" proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight-based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.

Attomole protein characterization by capillary electrophoresis-mass spectrometry.

Electrospray ionization with an ultralow flow rate (30 kilodaltons) at a resolving power of approximately 60,000 for injections of 0.7 x 10(-18) to 3 x 10(-18) mole of 8- to 29-kilodalton proteins with errors of <1 dalton in molecular mass. Using a crude isolate from human blood, a value of 28,780.6 daltons (calculated, 28,780.4 daltons) was measured for carbonic anhydrase, representing 1 percent by weight of the protein in a single red blood cell. Dissociation of molecular ions from 9 x 10(-18) mole of carbonic anhydrase gave nine sequence-specific fragment ions, more data than required for unique retrieval of this enzyme from the protein database.

Informatics and multiplexing of intact protein identification in bacteria and the archaea.

Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1-3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within approximately 5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at +/-0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectrometric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.

Two-dimensional mass spectrometry of biomolecules at the subfemtomole level.

Multiple dimensions of unique molecular structure information can now be obtained from proteins and DNA using mass spectrometry. Less than 10(-16) mol of the active major histocompatibility complex signaling peptide in a mixture of thousands can be identified. For large proteins (> 40 kDa), the high resolving power (> 10(5) and 10(-17) mol sensitivity of Fourier-transform mass spectrometry provide exact molecular weight values (+/- 1 or 2 Da) for mixture components, indicating error or modifications compared with the predicted DNA sequence. Selecting a specific molecular species, its two-dimensional spectrum indicates the part of the molecule that is modified; a three-dimensional spectrum of that fragment further isolates the modification site.

From primary structure to function: biological insights from large-molecule mass spectra.

As increasing information is available from genomic databases, mass spectrometry has begun to be used to identify and/or assess regions of predicted DNA or protein sequence. Mass spectrometry performance limits, together with experiments designed for genomic interplay, are being extended to allow accurate genotyping and protein profiling of cells at rates commensurate with the data-intensive future of biology.

Kinetics and regioselectivity of peptide-to-heterocycle conversions by microcin B17 synthetase.

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings introduced post-translationally in the 69 amino acid McbA gene product, an MccB17 precursor, by the microcin B,C,D enzyme complex. Both monocyclic and 4,2-bis-heterocyclic moieties are generated. The enzymatic cyclization involves 14 of the last 43 amino acids of McbA and requires the presence of the first 26 amino acids that function as a specificity-conferring propeptide. RESULTS: We have constructed maltose-binding protein (MBP)-McbA1-46 fusion proteins and have mutagenized the Gly39-Ser40-Cys41 (GSC) wild-type sequence to assess the regioselectivity and chemoselectivity of MccB17-synthetase-mediated heterocycle formation at the first two loci, residues 40 and 41 of McbA. Four single-site and four double-site substrates showed substantial differences in turnover as assessed by western assays, UV-visible spectroscopy and mass spectrometry. Cysteine-derived thiazoles form at a greater rate than serine-derived oxazoles. Formation of bis-heterocycles is sensitive both to composition and sequence context. CONCLUSIONS: The E. coli McbB,C,D MccB17 synthetase is the first peptide heterocyclization enzyme to be characterized. This study reveals substantial regioselectivity and chemoselectivity (thiazole > oxazole) at the most amino-terminal bis-heterocyclization site of McbA. The heterocyclization of GSS and GCC mutants of McbA1-46 by MccB17 synthetase demonstrates that the complex can efficiently generate tandem bis-oxazoles and bis-thiazoles, moieties not found in MccB17 but present in natural products such as hennoxazole and bleomycin. The observations suggest a common enzymatic mechanism for the formation of peptide-derived heterocyclic natural products.

In vivo processing and antibiotic activity of microcin B17 analogs with varying ring content and altered bisheterocyclic sites.

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings, and inhibits DNA gyrase. The role of individual and tandem pairs of heterocycles in bioactivity has not been determined previously. RESULTS: The two tandem 4,2-bisheterocycles in MccB17 were varied by expression of MccB17 or mutants containing altered sequences at Gly39-Ser40-Cys41 or Gly54-Cys55-Ser56. A mixture of five-nine-ring MccB17 isoforms were separated and quantitated for antibiotic potency. Mutagenesis of the thiazole-oxazole pair significantly affected antibiotic activity compared with the upstream oxazole-thiazole, which might stabilize partially cyclized intermediates against proteolysis. CONCLUSIONS: Enzymatic heterocyclization in native MccB17 occurs distributively. Antibiotic activity correlates with the number of rings and is differentially sensitive to both the location and the identity of the 4,2-tandem heterocycle pairs in MccB17. Such tandem heterocycles might be useful pharmacophores in combinatorial libraries.

Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization.

BACKGROUND: EntF is a 142 kDa four domain (condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N-acyl-serine trilactone siderophore enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. RESULTS: The Ser1138-->Cys mutant (kcat decreased 1000-fold compared with wild-type EntF) releases both enterobactin (75%) and linear (DHB-Ser)2 dimer (25%) as products. The His 1271-->Ala mutant (kcat decreased 10,000-fold compared with wild-type EntF) releases only enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser)2-O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. CONCLUSIONS: These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.

Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis.

A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry.

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.

Accurate base composition of double-strand DNA by mass spectrometry.

An accurate molecular weight (M r) assignment for a double-strand (ds) DNA determines or greatly restricts the possible number of each of its four bases, while the compositions for its two single-strand (ss) components can also be derived from their M r values. For a ds 64-mer (39 kDa), the ss-M r values (±0.5 Da) of its high-resolution mass spectrum from an electrospray ionization/Fourier transform instrument yield only the correct ds- and ss-base compositions. Literature mass spectra of lower mass accuracy show that such data can also restrict their possible composition assignments, with further discrimination using the abundance vs. base composition of small fragment ions from the dissociation of the ss molecular ions.

Thiaminase I (42 kDa) heterogeneity, sequence refinement, and active site location from high-resolution tandem mass spectrometry.

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B1). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×10(4). Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro(79) and Thr(177) in the 379 amino acid enzyme.

Complete large-molecule high-resolution mass spectra from 50-femtomole microvolume injection.

For electrospray ionization in Fourier-transform mass spectrometry, direct injection of 5×10(-14) mol (0.5 µL of 100 nM from a microvolume sample valve) of ubiquitin (8565 Da) into the flowing solvent stream yields a spectrum with 85:1 signal-to-noise ratio, 2-ppm mass accuracy, and isotopic resolution. Gated trapping for 100 µs from a 0.15-µL/min injection of 20-µM ubiquitin consumes 5×10(-18) mol, which produces a spectrum with 23:1 signal-to-noise ratio and τ;3×10(5) resolving power.

Fungal atopy in adult cystic fibrosis.

This study set out to estimate the prevalence of atopy to a variety of common ubiquitous fungi, including A. fumigatus, in cystic fibrosis (CF), and to evaluate the investigations by which the diagnosis was made. Particular attention was paid to the usefulness of skin testing and immunoassays in detecting which patients had simple fungal atopy, and which patients were at high risk of developing allergic bronchopulmonary mycoses. This cross-sectional study included 21 adult CF patients and 20 matched controls. Serum samples were taken for the measurement of total serum IgE and specific serum IgE to nine common fungi. Immediate hypersensitivity skin prick testing to each of the fungi was also performed. Simple fungal atopy was described in subjects fulfilling the following criteria: total serum IgE > 100 KU l(-1) with specific radioimmunoassay > or = grade 1 to at least one fungus and a positive skin prick test (SPT) > or = 3 mm to the same fungus. 'High risk' for developing allergic bronchopulmonary mycosis (ABPM) was described in subjects fulfilling the following criteria: total serum IgE > 200 KU l(-1) with specific radioimmunoassay > or = grade 2 to at least one fungus and a positive skin prick test (SPT) > or = 6 mm to the same fungus. The adult CF group had a significantly higher total SPT score (P=0.005) and mean total serum IgE (P<0.05) than controls. Forty-three percent of CF patients fulfilled the criteria for fungal atopy to at least a single fungus. Over half this group had an atopic tendency to more than one fungus. Nineteen percent of the CF group were at least 'high risk' of developing ABPM. Skin prick testing is a better marker of fungal atopy and a better predictor of those adult CF patients at higher risk of developing ABPM than specific radioimmunoassay serum testing. There is a high prevalence of fungal atopy in the adult CF population. Total serum IgE and skin prick testing are good predictors of fungal atopy and help predict those at risk of developing ABPM in CF.

Addition of epinephrine to epidural ropivacaine during labour--effects on onset and duration of action, efficacy, and systemic absorption of ropivacaine.

Addition of epinephrine to epidural anaesthetic solutions may enhance efficacy and duration of analgesia. We postulated that addition of epinephrine 5 microg.mL(-1) to epidural ropivacaine would improve efficacy, decrease systemic absorption and reduce neonatal effects. Twenty-one multiparous women were studied. An initial dose of ropivacaine 30 mg followed by an infusion of ropivacaine 10 mg.h(-1) was given via a lumbar epidural catheter. According to random allocation, epinephrine 5 microg.mL(-1) was added to ropivacaine. Ropivacaine concentrations were measured in maternal venous plasma after one hour of infusion and in both umbilical venous and maternal plasma at delivery. Neonatal neurologic and adaptive capacity score (NACS) tests were performed at 2 and 24 h postpartum. All women delivered vaginally. The groups had similar ropivacaine dose requirements, epidural-delivery intervals and satisfaction scores. Bromage scores for motor block were greater in the epinephrine group (2; range: 1-3) than controls (1; range: 0-2). Mean plasma ropivacaine concentrations (+/-SD) were less in the epinephrine group (0.17 +/- 0.05 mg.L(-1), n = 10) than controls (0.31 +/- 0.14 mg.L(-1), n = 11; P < 0.05) after one h of infusion but not at delivery. UV ropivacaine concentrations and NACS scores were similar in the two groups. The addition of epinephrine to ropivacaine decreases maternal plasma concentrations after one h of epidural infusion but also increases motor block.

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer.

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Absorbed radiation dose of the female breast during diagnostic multidetector chest CT and dose reduction with a tungsten-antimony composite breast shield: preliminary results.

AIM: To determine the absorbed radiation dose to the female breast during chest computed tomography (CT), and whether a custom-designed breast shield can reduce that dose. MATERIALS AND METHODS: Bilateral breast phantoms were combined with an anthropomorphic torso phantom. Each breast phantom contained 20 thermoluminescent dosimeter (TLD) cavities. Eight cavities were used per phantom. Absorbed radiation was measured using TLD 100 s. Three-stacked TLDs comprised a set. Three sets of three TLDs were positioned at eight designated locations and three depths (surface; 1 cm; 4 cm). One set of three TLDs was positioned at eight additional designations, 1cm deep. Each breast was divided anatomically into quadrants. In total, 32 TLD sets/96 TLDs were deployed. The breast-torso phantom was consecutively imaged using a 16-detector array CT machine. Subsequently, 32 new TLD sets were similarly placed, the phantom re-imaged in a likewise manner, but with the application of a tungsten-antimony composite breast shield. TLD readings were averaged and calculated. RESULTS: Average absorbed radiation doses for unshielded right and left breast phantoms ranged from 13.83-19.36 mGy, and 14-20.47 mGy, respectively. The absorbed dose in the shielded right and left breast was reduced to 6.64-8.12 mGy, and 6.7-8.03 mGy, respectively. Average absorbed radiation doses based on the depth for the unshielded breasts ranged from 15.4-18.3 mGy. Shielding reduced this dose to 7-7.9 mGy. Unshielded absorbed radiation doses based on anatomic quadrants ranged from 17.5-18.9 mGy. Shielding reduced this dose to 7-7.5 mGy. CONCLUSIONS: The average absorbed radiation dose to the unshielded female breast phantom is approximately 14-20 mGy. An externally applied shield can reduce this absorbed dose by 56-61%.