Comparison of the capabilities of liquid isoelectric focusing-one-dimensional nonporous silica reversed-phase liquid chromatography-electrospray ionization time-of-flight mass spectrometry and liquid isoelectric focusing-one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis mass mapping for the analysis of intact protein molecular masses.

Nonporous silica reversed-phase HPLC coupled to electrospray ionization with on-line time-of-flight mass spectrometric detection (NPS-RP-HPLC-ESI-TOF-MS) is shown to be an effective liquid phase method for obtaining the molecular masses of proteins from pH fractionated cellular lysates where the method is capable of generating the same banding patterns typically observed using gel phase one-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The liquid-phase mass spectrometry-based method provides a mass accuracy of at least 150 ppm, with 4000 mass resolution and provides improved sensitivity as the protein molecular mass (MW) decreases. The liquid and gel phase methods are shown to be complementary in terms of their mass range but the liquid phase method has the advantage over the gel method in that the analysis times are 50 times shorter, the mass accuracy is 70 times better and the resolution is 130 times higher. The liquid phase method is shown to be more effective for detection of proteins below 40 kDa, while the gel phase separation can access many more proteins, including more hydrophobic proteins, at increasing MW.

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line.

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.

Studies of posttranslational modifications in spiny dogfish myelin basic protein.

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.

An abnormal dipyridamole thallium/sestamibi fails to predict long-term cardiac events in vascular surgery patients.

Recent data demonstrate that dipyridamole-thallium (DTHAL) and sestamibi (DMIBI) are not predictive of adverse perioperative cardiac events in moderate-risk patients (one or more Eagle risk factors) undergoing major elective vascular surgery. Less data are available regarding the ability of DTHAL/DMIBI to predict adverse cardiac events on long term follow-up. We sought to determine whether an abnormal DTHAL/DMIBI is predictive of adverse cardiac events on long-term follow-up in moderate-risk patients undergoing major elective vascular surgery. Patients were enrolled prospectively between June 1997 and June 1999 at West Los Angeles VA and Harbor-UCLA Medical Centers. Adverse cardiac events were defined as congestive heart failure (CHF), myocardial infarction (MI), unstable angina (USA), and ventricular arrhythmias. Follow-up was obtained via clinic visits, telephone calls, and chart review. We studied 75 patients (76% male, 24% female) with a mean age of 65 years. Operative procedures were primarily femorodistal (83%) and aortic (16%). DTHAL/DMIBI results were normal in 35 patients (47%), demonstrated reversible ischemia in 26 (35%), and showed a fixed defect alone in 14 (18%). From the follow-up results of this study we conclude that there is no association between a reversible ischemia or an abnormal (fixed or reversible) DTHAL/DMIBI and adverse cardiac events or mortality on long-term follow-up in moderate-risk patients who have undergone major vascular surgery.

A simple model to help distinguish necrotizing fasciitis from nonnecrotizing soft tissue infection.

BACKGROUND: Necrotizing fasciitis (NF) has been associated with certain "hard" clinical signs (hypotension, crepitance, skin necrosis, bullae, and gas on x-ray), but these may not always be present. Using results of a previous study, we developed a simple model to serve as an adjunctive tool in diagnosing NF (admission WBC > 15.4 x 10(9)/L or serum sodium [Na] < 135 mmol/L) and determined its ability to distinguish between patients with NF and nonnecrotizing soft tissue infection (non-NF). STUDY DESIGN: A retrospective review was conducted of consecutive NF (n=31) and non-NF patients (n= 328) treated at a single institution during an 11-month period. Comparison of admission vital signs, physical examination findings, radiology results, and number of patients meeting model criteria was performed. RESULTS: Ninety percent of NF patients and 24% of non-NF patients met model criteria (p < 0.0001). The model had a sensitivity of 90%, a specificity of 76%, a positive predictive value of 26%, and a negative predictive value of 99% for diagnosing NF. Nineteen (61%) NF patients had no "hard" signs of NF; the model correctly classified 18 (95%) of these patients. CONCLUSIONS: Admission WBC greater than 15.4 x 10(9)/L and serum Na less than 135mmol/L are useful parameters that may help to distinguish NF from non-NF infection, particularly when classic "hard" signs of NF are absent.

Mouse liver selenium-binding protein decreased in abundance by peroxisome proliferators.

Several studies with two-dimensional gel electrophoresis (2-DE) have shown that the abundance of numerous mouse liver proteins is altered in response to treatment with chemicals known to cause peroxisome proliferation. The peptide masses from tryptic digests of two liver proteins showing dramatic decreases in abundance in response to numerous peroxisome proliferators were used to search sequence databases. The selenium-binding protein 2 (SBP2 formerly 56 kDa acetaminophen-binding protein, AP 56) and selenium-binding protein 1 (SBP1 formerly 56 kDa selenium-binding protein, SP 56) in mouse liver, proteins with a high degree of sequence similarity, were the highest ranked identities obtained. Identity with SBP2 was subsequently confirmed by immunodetection with specific antiserum. Treatment of mice with 0.025% ciprofibrate resulted in the more basic of this pair of proteins being decreased to 30% of control abundance while the acidic protein was decreased to 7% of the control amount. Dexamethasone treatment, in contrast, caused increases of 80% and 20% in the abundance of the acidic and basic forms, respectively. Administration of dexamethasone to mice in combination with ciprofibrate produced expression of the acidic SBP2 at 23% of the control level and the basic SBP2 at 36%, a slightly moderated reduction compared with the decrease that occurred with ciprofibrate alone. These data suggest that peroxisome proliferators such as ciprofibrate cause a decrease in the abundance of the SBP2, which leads to increased cell proliferation, even in the presence of an inhibitor such as dexamethasone. Such a decrease in SBP, thought to serve as cell growth regulation factors, could be central to the nongenotoxic carcinogenicity of the peroxisome proliferators observed in rodents.

Objective criteria may assist in distinguishing necrotizing fasciitis from nonnecrotizing soft tissue infection.

BACKGROUND: Optimal treatment of necrotizing fasciitis (NF) requires rapid diagnosis. The purpose of the study was to identify objective admission measurements that help differentiate NF from nonnecrotizing (non-NF) infection and, among NF patients, to identify admission factors that predict mortality. METHODS: Twenty-one NF cases were paired with matched non-NF controls. Statistical comparison of admission vital signs, laboratory values, and radiographic studies was performed. RESULTS: On multivariate analysis, admission white blood cell count (WBC) >14 x 10(9)/L, serum sodium <135 mmol/L, and blood urea nitrogen (BUN) >15 mg/dL separated NF from non-NF patients. Mortality for NF patients was predicted by admission WBC >30 x 10(9)/L. Mortality was also significantly increased for patients transferred from an outside institution prior to definitive therapy. CONCLUSIONS: Objective admission criteria (elevated WBC and BUN and decreased serum sodium) can assist in distinguishing NF from non-NF infections. The best objective predictor of mortality in NF patients is marked elevation of admission WBC.

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification.

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.

Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates.

A method for rapid profiling of water-soluble proteins from whole cell lysates has been developed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) following separation by reversed-phase high-performance liquid chromatography (RP HPLC). Rapid separation of proteins from cell lysates was achieved using columns packed with C18 nonporous (NP) silica beads. Using this method, the whole cell lysate water-soluble proteins of E. coli were separated in under 15 min. A method using two columns in series at different temperatures was used in order to provide high loadability without loss of separation efficiency. The nonporous packing in the columns provided for high recovery. Eluting fractions were collected and analyzed by MALDI-TOFMS to determine the molecular weights and peptide maps of the proteins. These methods provided for the rapid screening and identification of proteins from E. coli where the response of E. coli to L-arabinose induction was studied. In this work, it is demonstrated that NP RP HPLC with MALDI-TOFMS detection may serve as a rapid means of detecting and identifying changes in bacterial protein expression due to external stimuli.

Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to rapidly detect and profile large proteins from Escherichia coli whole cell lysates in the mass range 25-500 kDa. The bacterial samples were treated with guanidine hydrochloride and Triton X-100 to disrupt and solubilize the large inner membrane proteins. A sample preparation involving a nitrocellulose polymer film, and alpha-cyano-4-hydroxycinnamic acid, sinapinic acid or caffeic acid as matrix was utilized to rapidly monitor the presence of induced and repressed protein synthesis in response to L-arabinose catabolism in E. coli cells. The results were compared to those of 1-D or 2-D gel electrophoresis.

Effect of prehospital treatment on the outcome of status epilepticus in children.

Diazepam is administered to children in status epilepticus by paramedics in many Emergency Medical Services systems throughout the United States despite the lack of clear evidence that this therapy is safe and effective when employed in the prehospital environment. We reviewed the clinical course of 45 episodes of generalized convulsive status epilepticus (SE) in 38 children to determine the effect of prehospital diazepam therapy (given rectally or intravenously) on the clinical course of SE and subsequent patient management. Nineteen SE episodes were treated with prehospital diazepam therapy--9 episodes with rectal diazepam (mean dose: 0.6 mg/kg) and 10 episodes with intravenous diazepam (mean dose: 0.2 mg/kg). Prehospital diazepam therapy was associated with SE of shorter duration (32 min vs 60 min; P = .007) and a reduced likelihood of recurrent seizures in the emergency department (58% vs 85%; P = .045). There were no significant differences between rectal and intravenous diazepam therapy with regard to SE duration, intubation, or recurrent seizures in the emergency department. These data suggest that prehospital administration of diazepam may shorten the duration of SE in children and simplify the subsequent management of these patients in the emergency department.