Direct recording of high resolution mass spectra of gas chromatographic effluents. 1965.
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Gas Chromatography-Mass Spectrometry↗
Biomedical subjects
J T Watson
Publications and source records attributed to J T Watson.
Trapping of intermediates during the refolding of recombinant human epidermal growth factor (hEGF) by cyanylation, and subsequent structural elucidation by mass spectrometry.
Human epidermal growth factor (hEGF) contains 53 amino acids and three disulfide bonds. The unfolded, reduced hEGF is allowed to refold under mildly alkaline conditions. The folding is quenched at different time points by adjusting the pH to 3.0 with an acetic acid solution of 1-cyano-4-dimethylamino-pyridinium (CDAP) which traps folding intermediates via cyanylation of free sulfhydryl groups. The mixture of cyanylated intermediates is separated by reversed-phase HPLC; the fractions collected are identified by mass spectrometry. The disulfide structures of the intermediates are then determined by specific chemical cleavage and mass-mapping by MALDI-MS, a novel approach developed in our laboratory. The procedure of quenching and trapping of disulfide intermediates in acidic solution minimizes sulfhydryl-disulfide exchange, and therefore provides a good measure of folding kinetics and preservation of intermediate species. Our cyanylation methodology for disulfide mapping is simpler, faster, and more sensitive than the more conventional approach. Among 18 folding intermediates isolated and identified at different time points, disulfide structures of seven well-populated intermediates, including two non-native isomers with scrambled disulfide structures, one 2-disulfide intermediate, and four 1-disulfide intermediates, have been characterized; most of them possess non-native disulfide structures.
Amino Acid Sequence↗
Leg kinematics and muscle activity during treadmill running in the cockroach, Blaberus discoidalis: I. Slow running.
We have combined high-speed video motion analysis of leg movements with electromyogram (EMG) recordings from leg muscles in cockroaches running on a treadmill. The mesothoracic (T2) and metathoracic (T3) legs have different kinematics. While in each leg the coxa-femur (CF) joint moves in unison with the femurtibia (FT) joint, the relative joint excursions differ between T2 and T3 legs. In T3 legs, the two joints move through approximately the same excursion. In T2 legs, the FT joint moves through a narrower range of angles than the CF joint. In spite of these differences in motion, no differences between the T2 and T3 legs were seen in timing or qualitative patterns of depressor coxa and extensor tibia activity. The average firing frequencies of slow depressor coxa (Ds) and slow extensor tibia (SETi) motor neurons are directly proportional to the average angular velocity of their joints during stance. The average Ds and SETi firing frequency appears to be modulated on a cycle-by-cycle basis to control running speed and orientation. In contrast, while the frequency variations within Ds and SETi bursts were consistent across cycles, the variations within each burst did not parallel variations in the velocity of the relevant joints.
Animals↗
Leg kinematics and muscle activity during treadmill running in the cockroach, Blaberus discoidalis: II. Fast running.
We have combined kinematic and electromyogram (EMG) analysis of running Blaberus discoidalis to examine how middle and hind leg kinematics vary with running speed and how the fast depressor coxa (Df) and fast extensor tibia (FETi) motor neurons affect kinematic parameters. In the range 2.5-10 Hz, B. discoidalis increases step frequency by altering the joint velocity and by reducing the time required for the transition from flexion to extension. For both Df and FETi the timing of recruitment coincides with the maximal frequency seen for the respective slow motor neurons. Df is first recruited at the beginning of coxa-femur (CF) extension. FETi is recruited in the latter half of femur-tibia (FT) extension during stance. Single muscle potentials produced by these fast motor neurons do not have pronounced effects on joint angular velocity during running. The transition from CF flexion to extension was abbreviated in those cycles with a Df potential occurring during the transition. One effect of Df activity during running may be to phase shift the beginning of joint extension so that the transition is sharpened. FETi is associated with greater FT extension at higher running speeds and may be necessary to overcome high joint torques at extended FT joint angles.
Animals↗
Long-term mechanical circulatory support system reliability recommendation: American Society for Artificial Internal Organs and The Society of Thoracic Surgeons: long-term mechanical circulatory support system reliability recommendation.
Jointly developed by members of the American Society for Artificial Internal Organs and the Society of Thoracic Surgeons along with staff from the Food and Drug Administration, the National Heart, Lung and Blood Institute and other experts, this recommendation describes the reliability considerations and goals for Investigational Device Exemption and Premarket Approval submissions for long-term, mechanical circulatory support systems. The recommendation includes a definition of system failure, a discussion of an appropriate reliability model, a suggested in vitro reliability test plan, reliability considerations for animal implantation tests, in vitro and animal in vivo performance goals, the qualification of design changes during the Investigational Device Exemption clinical trial, the development of a Failure Modes Effects and Criticality Analysis, and the reliability information for surgeons and patient candidates. The document will be periodically reviewed to assess its timeliness and appropriateness within five years.
Animals↗
Treatment of complex lateral plateau fractures using Ilizarov techniques.
Fourteen high energy atypical Schatzker Types I and II fractures were treated using a combination of contemporary internal fixation techniques and Ilizarov methodologies. Fracture were atypical if the primary lateral condylar fracture line extended anteromedially with complete detachment of the tibial tubercle, or posteromedially with extension into the medial or posterior medial tibial condyle, both in association with comminution of the lateral condyle and impaction of the lateral articular surface. Preoperative traction computed tomography scans were used to direct incisions to the area of joint involvement. Fixation included hook plate stabilization of anterior tubercle fragments in five fractures. Adjunctive lateral, medial, or posterior antiglide plates were used where olive wire stabilization was contraindicated because of anatomic constraints. Fractures were neutralized by a three-ring Ilizarov external fixator. At followup, average 19.2 months (range, 8-67 months), all fractures had healed, and 85% had good or excellent knee scores (Knee Society clinical rating scale). Five patients had minor pin tract complications and one patient had a superficial wound slough. This combined approach has shown excellent results for this complex fracture pattern without the severe soft tissue complications associated with internal fixation techniques for high energy fractures.
Adult↗
Outcomes following repair of quadriceps tendon ruptures.
OBJECTIVES: The purpose of this study was to examine critically the outcomes of patients sustaining a quadriceps tendon rupture and to compare outcomes in patients with bilateral simultaneous ruptures versus a unilateral rupture. DESIGN: Retrospective review. SETTING: Patients were treated at a Level I trauma center. PATIENTS/PARTICIPANTS: Fifty-one quadriceps tendon ruptures in thirty-nine patients were evaluated. A mean four-year follow-up (range 13 to 204 months) was available for forty-eight tendon ruptures. INTERVENTIONS: All patients except one were treated with operative repair of the quadriceps tendon rupture(s). MAIN OUTCOME MEASUREMENTS: Patients were assessed by physical examination, Lysholm and Tegner scores, a functional questionnaire, quadriceps isokinetic testing, and radiographs. RESULTS: A statistically greater number of patients in the bilateral simultaneous rupture group had a systemic illness associated with tendon rupture (p = 0.014). This result did not adversely affect outcome as compared with patients with unilateral ruptures. Mean range of motion was 123 degrees in injured knees. Eighty-four percent of working patients returned to their previous occupations. More than half the patients, however, in general the most active, could no longer participate in their preinjury recreational activities. Fifty-three percent of unilateral rupture patients had persistent quadriceps strength deficits (>20 percent) in the injured extremity. Both quadriceps and hamstring isokinetic testing correlated significantly with Lysholm and Tegner scores. CONCLUSIONS: Most patients with bilateral simultaneous and unilateral tendon repairs can expect a good range of motion and return to their previous occupation, but many have persistent weakness and difficulty returning to higher level sporting activities.
Activities of Daily Living↗
Cardiac MR imaging: report of a working group sponsored by the National Heart, Lung, and Blood Institute.
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Cardiovascular Diseases↗
Comparison of the compression hip screw with the Medoff sliding plate for intertrochanteric fractures.
The Medoff sliding plate was designed to achieve compression along the femoral neck and the longitudinal axis of the femoral shaft theoretically to improve the treatment of intertrochanteric hip fractures. The Medoff sliding plate was compared with a standard compression hip screw in a randomized, prospective study for the fixation of 160 stable and unstable intertrochanteric fractures with an average followup of 9.5 months (range, 6-26 months). Overall, 91 fractures were treated using the compression hip screw and 69 were treated with the Medoff sliding plate. Stable fracture patterns (46) united without complication in both treatment groups. Unstable fractures (114) had an overall failure rate of 9.6%, 14% (nine patients) with the compression hip screw and 3% (two patients) with the Medoff plate; this difference was significantly different. The time to union for the 114 unstable fractures was not significantly different between the two devices. For all patients, no differences in lengths of hospitalization, return to ambulatory status before fracture, postoperative living status, or postoperative pain was observed between the two device groups. Use of the Medoff plate for all fracture types was associated with a significantly higher amount of blood loss and operating time.
Activities of Daily Living↗
A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry.
A highly activated ester containing a fixed positive charge, S-pentafluorophenyl [tris(2,4,6-trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide), has been synthesized as a reagent for N-terminal modification of peptides. Stable in aqueous acetonitrile solution during extended storage, TMPP-AcSC6F5 bromide reacts with unprotected peptides through p-(dimethylamino)pyridine (DMAP)-promoted amidation in aqueous acetonitrile (15 min, ambient temperature) to form N-TMPP-Ac derivatives of peptides. These peptide derivatives are readily amenable to analysis by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Greater than 90% conversion has been observed in transforming low-nanomole quantities of analyte using molar ratios of 1:5:10 (peptide/reagent/ DMAP). For reactions at the picomole level a slightly modified stoichiometry, with molar ratios of 1:10:500, is employed. Owing to the high reaction efficiency and the tolerance to moderate excess reagent and base during analysis by FAB- and MALDI-MS, the reaction mixture containing the modified peptides can be analyzed directly in most cases, without sample cleanup. Examples of the preparation and analysis of a variety of N-TMPP-acetyl-peptides (TMPP-Ac-peptides) ranging from hexamers to 15-mers are given. Collisionally activated dissociation tandem mass spectrometry of TMPP-Ac-derivatives showed dominant a-type ions, accompanied by d- and c-type ions in some cases, allowing sequence determination to be made in a straightforward manner.
Amino Acid Sequence↗
A novel methodology for assignment of disulfide bond pairings in proteins.
A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.
Chromatography, High Pressure Liquid↗
Separation and quantitation of linoleic acid oxidation products in mammary gland tissue from mice fed low- and high-fat diets.
We have developed an assay for the isolation and quantitation by gas chromatography-mass spectrometry (GC-MS) of free 9- and 13-hydroxyoctadecadienoic acid (9-HODE, 13-HODE) in the mammary glands of female mice. Internal standards consisting of 18O2-labeled analogs of 9- and 13-HODE are added to pulverized frozen tissue prior to extraction with ethanol. Nonlipid materials are removed in a chloroform/methanol/water step. The remaining lipid material is methylated with ethereal diazomethane, and much of the nonoxygenated fatty acid methyl esters are removed via silica solid-phase extraction. Samples are either further derivatized with bis(trimethylsilyl)trifluoroacetamide to form the trimethylsilyl ethers for quantitative analysis by GC-MS or are analyzed as the methyl esters by chiral high-performance liquid chromatography to determine the enantiomeric distribution of the 9- and 13-HODE. The extraction and quantitation protocol was applied to the analysis of mammary glands for free 9- and 13-HODE from mice fed isocaloric diets containing 20% corn oil, 5% corn oil, or 20% beef tallow. Chiral analysis of the products showed higher production of 13(S)-HODE relative to 13(R)-HODE; the enantiomeric excess is most likely due to enzymatic production of 13-HODE superimposed on a background of autoxidative production of 13(R)- plus 9(S)- and 9(R)-HODE. In addition, the effect of sample handling and storage conditions on the formation of 9- and 13-HODE in the samples was assessed by exposing aliquots of a common pool of rat mammary gland tissue to specified conditions prior to analysis. This methodology will be important during investigations of the contribution of linoleate oxidation products to the enhancement of mammary tumorigenesis by dietary fat.
Analysis of Variance↗
Early complications in the operative treatment of patella fractures.
OBJECTIVE: To identify and review early complications in the operative treatment of patella fractures. DESIGN: Retrospective review. SETTING: Single tertiary care institution with multiple surgeons, including generalists and fellowship trained subspecialists. PATIENTS: A consecutive series of eighty-seven patella fractures over a five year period was reviewed. Patients treated nonoperatively or with partial or total patellectomy were excluded. Minimum follow-up to fracture healing (four months) was available in fifty-one fractures. INTERVENTION: Modified tension band wire fixation was used in forty-nine fractures, whereas two fractures were treated with tension band wires threaded through cannulated screws. OUTCOME MEASURES: Early complications such as loss of reduction or fixation, infection, or soft-tissue problems were evaluated. RESULTS: Displacement of > or = 2 mm before healing was noted in eleven fractures. The displacement could be attributed to technical errors in five cases, and to patient noncompliance with postoperative activity restrictions in another five cases. Two cases of superficial infection were documented. Nine patients with symptomatic hardware required hardware removal. CONCLUSIONS: Twenty-two percent of fractures treated with tension band wiring and early motion displaced > or = 2 mm within the early postoperative period. Technical errors or patient noncompliance were identified as factors. The incidence of early complications in operatively treated patella fractures is higher than previously reported.
Adolescent↗
Intramedullary nailing of unstable diaphyseal fractures of the tibia with distal intraarticular involvement.
OBJECTIVE: To evaluate the efficacy of intramedullary nailing in diaphyseal tibia fractures with distal intraarticular involvement. DESIGN: Retrospective. SETTING: Henry Ford Hospital, a level I trauma center. PATIENTS/PARTICIPANTS: Twenty patients with twenty fractures at an average of twenty-two months of follow-up were evaluated. There were fifteen closed and five open fractures. INTERVENTION: All fractures were stabilized with lag screw fixation (with or without supplemental plates) of the intraarticular-fracture extension or ankle fracture, and intramedullary nailing of the diaphyseal tibia fracture. MAIN OUTCOME MEASUREMENTS: Time to bony union, malunion, knee and ankle range of motion, early arthrosis, and any complications of treatment were assessed. RESULTS: Nineteen fractures healed, with an average time to bony union of seventeen weeks. One nonunion after a grade IIIB open fracture required exchange nailing and healed after sixty-two weeks. Nineteen fractures had excellent alignment after healing. There were no infections. CONCLUSIONS: The indications for intramedullary nailing of unstable diaphyseal tibia fractures may be extended to include certain fractures with distal extension into the ankle joint, as well in a tibial shaft fracture occurring in combination with a noncontiguous ipsilateral ankle fracture.
Adult↗
A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols. The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS. The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions. Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups. Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes.
Cysteine↗
Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)- octadecatrienoic acids from alpha-linolenic acid.
Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1 and hPGHS-2) were expressed by transient transfection of COS-1 cells. Microsomes prepared from the transfected cells were used to measure the rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty acid substrates including eicosapentaenoic, arachidonic, dihomo-gamma-linolenic > alpha-linolenic (delta 9, 12, 15), gamma-linolenic, and linoleic acids. Comparisons of kcat/Km values indicate that the order of efficiency of oxygenation is arachidonate > dihomo-gamma-linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-linolenate was a particularly poor substrate for hPGHS-1. Gamma-Linolenate and eicosapentaenoate were poor substrates for both isozymes, but in each case, these two fatty acids were better substrates for hPGHS-2 than hPGHS-1. These studies of substrate specificities are consistent with previous studies of the interactions of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have indicated that the cyclooxygenase active site of PGHS-2 is somewhat larger and more accommodating than that of PGHS-1. The major products formed from linoleate and alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-linolenate is positioned in the cyclooxygenase active site with a kink in the carbon chain such that hydrogen abstraction occurs from the omega 5-position in contrast to abstraction of the omega 8-hydrogen from other substrates.
Animals↗
Dual pathways for tactile sensory information to thoracic interneurons in the cockroach.
The escape system of the American cockroach is both fast and directional. In response to wind stimulation both of these characteristics are largely due to the properties of the ventral giant interneurons (vGIs), which conduct sensory information from the cerci on the rear of the animal to type A thoracic interneurons (TIAs) in the thoracic ganglia. The cockroach also escapes from tactile stimuli, and although vGIs are not involved in tactile-mediated escapes, the same thoracic interneurons process tactile sensory information. The response of TIAs to tactile information is typically biphasic. A rapid initial depolarization is followed by a longer latency depolarization that encodes most if not all of the directional information in the tactile stimulus. We report here that the biphasic response of TIAs to tactile stimulation is caused by two separate conducting pathways from the point of stimulation to the thoracic ganglia. Phase 1 is generated by mechanical conduction along the animal's body cuticle or other physical structures. It cannot be eliminated by complete lesion of the nerve cord, and it is not evoked in response to electrical stimulation of abdominal nerves that contain the axons of sensory receptors in abdominal segments. However, it can be eliminated by lesioning the abdominal nerve cord and nerve 7 of the metathoracic ganglion together, suggesting that the relevant sensory structures send axons in nerve 7 and abdominal nerves of anterior abdominal ganglia. Phase 2 of the TIA tactile response is generated by a typical neural pathway that includes mechanoreceptors in each abdominal segment, which project to interneurons with axons in either abdominal connective. Those interneurons with inputs from receptors that are ipsilateral to their axon have a greater influence on TIAs than those that receive inputs from the contralateral side. The phase 1 response has an important role in reducing initiation time for the escape response. Animals in which the phase 2 pathway has been eliminated by lesion of the abdominal nerve cord are still capable of generating a partial startle response with a typically short latency even when stimulated posterior to the lesion.
Animals↗
Matrix-assisted laser desorption ionization mass spectrometry: applications in peptide and protein characterization.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has quickly gained notoriety as an analytical tool that can accurately determined the molecular weight of proteins up to (and exceeding) 100 kDa from low-picomole amounts of sample. Beyond simple molecular weight determinations, investigators are developing this mass spectrometric technique to obtain more structural information from proteins using a combination of chemical and enzymatic modifications of the analyte prior to analysis by MALDI-MS. Covalent post-translational modifications such as phosphorylation and disulfide bond formation can be identified and localized. MALDI-MS also can be used to rapidly mass-map protein digests for sequence confirmation, often without requiring peptide fractionation. The potential for direct sequencing of peptides and proteins of unknown structure has been successfully demonstrated using several different MALDI-MS approaches. The objective of this report is to introduce MALDI-MS to the non-mass-spectrometrist and to describe applications of this new analytical technique for acquiring information related to peptide and protein structure.
Amino Acid Sequence↗