Advances in proteome analysis by mass spectrometry.
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Mass Spectrometry↗
Biomedical subjects
T J Griffin
Publications and source records attributed to T J Griffin.
Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.
We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.
Fungal Proteins↗
Advances in proteome analysis by mass spectrometry.
Proteome characterization using mass spectrometry is essential for the systematic investigation of biological systems and for the study of gene function. Recent advances in this multifaceted field have occurred in four general areas: protein and peptide separation methodologies; selective labeling chemistries for quantitative measurement of peptide and protein abundances; characterization of post-translational protein modifications; and instrumentation.
Chromatography, High Pressure Liquid↗
Toward a high-throughput approach to quantitative proteomic analysis: expression-dependent protein identification by mass spectrometry.
The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.
Autoanalysis↗
In vitro transposition system for efficient generation of random mutants of Campylobacter jejuni.
Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.
Base Sequence↗
Genetic identification by mass spectrometric analysis of single-nucleotide polymorphisms: ternary encoding of genotypes.
An approach to genetic identification using biallelic single-nucleotide polymorphism (SNP) genetic markers is described in which the three possible genotypes, AA, Aa, or aa, where "A" and "a" represent the two SNP alleles, are assigned a ternary (base 3) digit of 0, 1, or 2, respectively. Genotyping an individual over a panel of separate SNP markers produces a composite ternary genetic code that can be converted to an easily stored, decimal (base 10) genetic identification number. The unambiguous identification of 11 individuals is demonstrated using ternary genetic codes generated from MALDI-TOF mass spectrometric genotyping data from 7 different SNP markers.
Alleles↗
Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry.
Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.
Genotype↗
Comparison of a 5 day regimen of cefdinir with a 10 day regimen of cefprozil for treatment of acute exacerbations of chronic bronchitis.
Patients with acute exacerbations of chronic bronchitis were treated with cefdinir 300 mg bd for 5 days or cefprozil 500 mg bd for 10 days in a prospective, randomized, double-blind, multicentre study. Of the 548 patients enrolled, 281 (51%) were evaluable. The clinical cure rates at the test-of-cure visit were 80% (114/142) and 72% (100/139) for the evaluable patients treated with cefdinir and cefprozil, respectively. Respiratory tract pathogens were isolated from 409 (75%) of 548 admission sputum specimens, with the predominant pathogens being Haemophilus parainfluenzae, Haemophilus influenzae, Staphylococcus aureus and Moraxella catarrhalis. The microbiological eradication rates at the test-of-cure visit were 81% (157 of 193 pathogens) and 84% (166 of 198 pathogens) for the evaluable patients treated with cefdinir and cefprozil, respectively. Adverse event rates while on treatment were equivalent between the two treatment groups. The incidence of diarrhoea during therapy was higher for patients treated with cefdinir (17%) than for patients treated with cefprozil (6%) (P < 0.01), but most cases were mild and did not lead to discontinuation of treatment. These results indicate that a 5 day regimen of cefdinir is as effective and safe in the treatment of patients with acute exacerbations of chronic bronchitis as a 10 day regimen of cefprozil.
Acute Disease↗
A comparison of a five-day regimen of cefdinir with a seven-day regimen of loracarbef for the treatment of acute exacerbations of chronic bronchitis.
To compare the efficacy and safety of five-day cefdinir treatment with seven-day loracarbef treatment in patients with acute exacerbations of chronic bronchitis, 586 patients were enrolled in a multicentre, randomised, double-blind trial. Patients received either five days of treatment with cefdinir (n = 291) at 300 mg twice daily or seven days of treatment with loracarbef (n = 295) at 400 mg twice daily. Microbiological assessments were done on sputum specimens obtained at admission and at the two post-therapy visits, if available. The clinical cure rates were 86% (138/160) and 85% (141/166) for the evaluable patients treated with cefdinir and loracarbef, respectively. Respiratory tract pathogens were isolated from 457 (78%) of 586 admission sputum specimens, with the predominant pathogens being Haemophilus parainfluenzae, H. influenzae, Moraxella catarrhalis and Staphylococcus aureus. The microbiological eradication rates at the test-of-cure visit were 88% (193/219 pathogens) and 90% (227/251 pathogens) for the evaluable patients treated with cefdinir and loracarbef, respectively. Adverse event rates while on treatment were 30% and 21% for cefdinir- and loracarbef-treated patients, respectively. These results indicate that a five-day regimen of cefdinir is effective and safe for the treatment of patients with acute exacerbations of chronic bronchitis.
Adolescent↗
In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.
We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.
DNA Transposable Elements↗
Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry.
An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.
Base Sequence↗
Tn552 transposase catalyzes concerted strand transfer in vitro.
The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg2+. Although it stimulates strand transfer overall, Mn2+ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.
Base Sequence↗
An approach to predicting the stabilities of peptide nucleic acid:DNA duplexes.
An approach is described for predicting peptide nucleic acid (PNA):DNA duplex stability from base sequence by approximating the total free energy of dissociation, delta G degree tot, for these duplexes as the sum of five parameters: (a) a nearest-neighbor interaction summation term, sigma Nj delta G degree j; (b) an initiation term, delta G degree i; (c) a dangling-end stabilization term, delta G degree e; (d) a PNA:DNA stabilization term per nearest-neighbor interaction, delta G degree pna; and (e) an ionic strength term, delta G degree Na. Parameters (a) and (b) are approximated using previously determined values for DNA:DNA duplexes, whereas parameters (c), (d), and (e) are empirically determined. These terms are used to calculated delta G degree tot, which is used in conjunction with a transition enthalpy (delta H degree) value, also approximated from nearest-neighbor values previously derived for DNA:DNA duplexes, to calculate a melting temperature (Tm) for the PNA:DNA duplex. Predicted Tm values calculated by this approach agreed fairly well with measured values for 11 different PNA:DNA duplexes, as well as with literature values. The approach also accurately models ionic strength effects.
Base Sequence↗
Comparative therapeutic efficacy of clinafloxacin in a Pseudomonas aeruginosa mouse renal abscess model.
A deep-seated Pseudomonas aeruginosa mouse kidney abscess model was used to compare the therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, with that of clinically relevant standard drugs. Following 50 mg/kg oral doses, twice daily for five consecutive days, clinafloxacin produced a 4 log decrease in mean bacterial count, the greatest decrease of all drugs tested. The same dosage regimen resulted in complete bacterial eradication in 88% of the kidneys. No other compound produced total bacterial clearance in 50% of the kidneys at the highest dose tested.
Abscess↗
Genetic analysis by peptide nucleic acid affinity MALDI-TOF mass spectrometry.
The ability to analyze multiple polymorphic sites rapidly and accurately is crucial in all areas of genetic analysis. We have developed an approach for the detection of multiple point mutations, using allele-specific, mass-labeled, peptide nucleic acid (PNA) hybridization probes, and direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The composite mass spectra produced contain peaks of distinct masses corresponding to each allele present, resulting in a mass spectral "fingerprint" for each DNA sample. The hybridization characteristics of PNA:DNA duplexes were found to be highly dependent on both base content and sequence. Results from the analysis of four polymorphic sites contained in exon 4 of the human tyrosinase gene show that this approach is simple, rapid, and accurate with potential applications in many areas of genetic analysis.
Alleles↗
Comparison of cefdinir and cefaclor in treatment of community-acquired pneumonia.
Six hundred ninety patients were enrolled in a multicenter, randomized, double-blind trial comparing the efficacy and safety of cefdinir with those of cefaclor in the treatment of community-acquired pneumonia. Patients received either 10 days of treatment with cefdinir (n = 347) at 300 mg twice daily or 10 days of treatment with cefaclor (n = 343) at 500 mg three times daily. Microbiological assessments were performed on sputum specimens obtained at admission and at the two posttherapy visits, if available. Respiratory tract pathogens were isolated from 538 (78%) of 690 patient admission sputum specimens, with the predominant pathogens being Haemophilus parainfluenzae, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. The microbiological eradication rates at the test-of-cure visit were 92% (238 of 260 pathogens) and 93% (245 of 264 pathogens) for the evaluable patients treated with cefdinir and cefaclor, respectively. A satisfactory clinical response (cure plus improvement) was achieved in 89% (166 of 187) and 86% (160 of 186) of the evaluable patients treated with cefdinir and cefaclor, respectively. Except for the incidence of diarrhea, adverse event rates while on treatment were equivalent between the two treatment groups. Diarrhea incidence during therapy was higher for patients treated with cefdinir (13.7%) than for patients treated with cefaclor (5.3%). These results indicate that cefdinir is effective and safe in the treatment of patients with pneumonia.
Adolescent↗
A dentofacial deformity associated with incontinentia pigmenti: report of a case.
A case of IP in a 16-year-old girl has been presented. This patient manifested classic ectodermal and mesodermal anomalies. We present this case to illustrate a rare etiologic factor in the development of dentofacial deformities that can be treated in the conventional manner.
Adolescent↗
Purification and preliminary characterization of the Escherichia coli K-12 recF protein.
The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair. To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product. The RecF protein was overproduced and purified to apparent homogeneity. N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present. The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays. Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM. The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity. Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.
Amino Acid Sequence↗