Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry.

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.

Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer.

Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI-FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.

Numerical simulation of ion transport in an atmosphere-to-vacuum interface taking into account gas dynamics and space charge.

A two-step approach was developed for the study of ion transport in an atmospheric pressure interface. In the first step, the flow in the interface was numerically simulated using the standard gas dynamic package ANSYS CFX 15.0. In the second step, the calculated fields of pressure, temperature, and velocity were imported into a custom-built software application for simulation of ion motion under the influence of both gas dynamic and electrostatic forces. To account for space charge effects in axially symmetric interfaces an analytical expression was used for the Coulomb force. For all other types of interfaces, an iterative approach for the Coulomb force computation was developed. The simulations show that the influence of the space charge is the main contributor to the loss of ion current in the heated capillary. In addition, the maximum ion current which can be transmitted through the heated capillary (0.58 mm inner diameter and 58.5 mm length) is limited to ∼6 nA for ions with m/z = 508 Da and with reduced ion mobility 1.05 cm2V-1s-1. This limit remains practically constant and independent of the ion current at the entrance of the capillary. For a particular ion type, this limit depends on its m/z ratio and ion mobility.

Advancement of atmospheric-vacuum interfaces for mass spectrometers with a focus on increasing gas throughput for improving sensitivity.

Ion sampling from an electrospray ionization (ESI) source was improved by increasing gas conductance of the MS inlet by 4.3-fold. Converting the gas throughput (Q) into sensitivity improvement was dependent on ion desolvation and handling of the gas load. Desolvation was addressed by using a novel slot shaped inlet that exhibited desolvation properties identical to the 0.58 mm i.d capillary. An assay tailored for "small molecules" at high chromatographic flow rate (500 µL/min) yielded a compound dependent 6.5 to 14-fold signal gain while analysis at nano chromatographic flow rate (300 nL/min) showed 2 to 3.5-fold improvement for doubly charged peptides. Improvement exceeding the Q (4.3-fold) at high chromatographic flow rate was explained by superior sampling of the spatially dispersed ion spray when using the slot shaped capillary. Sensitivity improvement across a wide range of chromatographic flow rate confirmed no compromise in ion desolvation with the increase in Q. Another improvement included less overflow of gas into the mass analyzer from the foreline region owing to the slot shape of the capillary. By doubling the roughing pump capacity and operating the electrodynamic ion funnel (EDIF) at ∼4 Torr, a single pumping stage was sufficient to handle the gas load. The transport of solvent clusters from the LC effluent into the mass analyzer was prevented by a "wavy shaped" transfer quadrupole and was compared with a benchmark approach that delivered ions orthogonally into a differentially pumped dual EDIF at comparable gas Q.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

BACKGROUND: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. RESULTS: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h. CONCLUSIONS: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3-5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.

Chick embryo pancreatic transplants reverse experimental diabetes of rats.

The effectiveness of xenogeneic embryonic tissue in the treatment of experimental diabetes has been investigated in rats. The splenic lobes (80) of 15- to 18-d-old chick embryos, composed almost exclusively of endocrine tissue, were implanted directly into the hepatic parenchyma of the rat recipient. The biochemical and metabolic changes in the recipients suggest that embryonic transplants of 15-d-old chick pancreases were able to significantly improve, for a prolonged period of time (18 mo), the diabetic state of nonimmunosuppressed rats. None of the recipients of 18-d-old embryos splenic lobes exhibited a long-term improvement of the diabetic state after transplantation. The complete destruction of the pancreatic B cells of the recipients was assessed by: (a) immunocytochemical investigations of the recipient's pancreas, (b) measurement of insulin in the liver and pancreas of the recipients and (c) in situ vascular perfusion of their pancreas submitted to high glucose challenge. The results suggest that pancreatic tissue of the 15-d-old embryos is immunologically immature lacking one or several lymphocyte subsets implicated in the afferent lood of "non-self" recognition.

Age-Related Deterioration of Perineuronal Nets in the Primary Auditory Cortex of Mice.

Age-related changes in inhibitory neurotransmission in sensory cortex may underlie deficits in sensory function. Perineuronal nets (PNNs) are extracellular matrix components that ensheath some inhibitory neurons, particularly parvalbumin positive (PV+) interneurons. PNNs may protect PV+ cells from oxidative stress and help establish their rapid spiking properties. Although PNN expression has been well characterized during development, possible changes in aging sensory cortex have not been investigated. Here we tested the hypothesis that PNN+, PV+ and PV/PNN co-localized cell densities decline with age in the primary auditory cortex (A1). This hypothesis was tested using immunohistochemistry in two strains of mice (C57BL/6 and CBA/CaJ) with different susceptibility to age-related hearing loss and at three different age ranges (1-3, 6-8 and 14-24 months old). We report that PNN+ and PV/PNN co-localized cell densities decline significantly with age in A1 in both mouse strains. In the PNN+ cells that remain in the old group, the intensity of PNN staining is reduced in the C57 strain, but not the CBA strain. PV+ cell density also declines only in the C57, but not the CBA, mouse suggesting a potential exacerbation of age-effects by hearing loss in the PV/PNN system. Taken together, these data suggest that PNN deterioration may be a key component of altered inhibition in the aging sensory cortex, that may lead to altered synaptic function, susceptibility to oxidative stress and processing deficits.

A freeze-fracture and thin section study of the interaction of blood platelets with native type I collagen fibrils.

The membranes of non-activated and activated human blood platelets have been studied with freeze-fracture and conventional electron microscopy. Aggregated platelets activated by ADP or by native collagen fibrils did not show any intramembrane particle clustering. No intramembrane modification is detectable at the contact regions with collagen fibrils. However, a local densification of the cortical cytoplasm is noted in thin sections where platelets make contact with collagen fibrils. These results suggest that either the membrane receptor for collagen is not visualized by freeze-fracturing or that, as suggested previously, the binding site on collagen may not have a specificity and affinity as high as expected from a conventional receptor-ligand interaction.

Ultrastructural immunolocalization of elastic fibers in rat blood vessels using the protein A-gold technique.

Identification of elastic fibers at the ultrastructural level is accomplished by a post-embedding immunohistochemical technique using the protein A-colloidal gold method. Antisera against elastins from human dermis and rat aorta have been characterized by radioimmunoassay and then applied to thin sections of rat blood vessels. Two fixative solutions and two embedding media have been tested. Both antibodies bind to elastic fibers of normal arteries and veins, indicating crossreactions among organs and species. The high sensitivity of this method is demonstrated by its application to the detection of neo-elastogenesis in the intimal thickening of aortic grafts.

Evaluation of histamine release following intravenous injection of ionic and nonionic contrast media.

To evaluate histamine release (HR) following injection of radiocontrast media and to test the predictive value of peak-expiratory-flow rate (PEFR) in detecting high-risk patients, the authors performed in two series 90 intravenous pyelograms (IVPs). In the first group, HR was measured by a fluorometric method after injection of Hexabrix (25 patients) and Iopamiron (25 patients). In the second group, HR measured by a radioimmunoassay, and PEFR measured by a peak flowmeter were investigated after injection of Hexabrix (10 patients), Telebrix (10 patients), Omnipaque (10 patients) and Iopamiron (10 patients). Histamine release in groups 1 and 2, and PEFR in group 2, were not significantly modified by the injection of each radiocontrast media. For the four patients (two per group) who experienced minor allergic side effects, the levels of HR and PEFR were always within the normal ranges.

Effect of iodinated contrast media on blood clotting.

Recently, blood clot formation in catheters used for the injection of nonionic contrast media (CM) during angiography has been reported as being due to activation of hemostasis in the catheter. However, CM exhibit inhibitory properties regarding coagulation and platelet functions. The effect on blood clotting of iohexol, iopamidol, ioxaglate, diatrizoate, and ioxitalamate at a ratio of 10% v/v with nonanticoagulated human whole blood was evaluated using the kinetics of fibrinopeptide A (FpA) generation. Blood aliquots were taken every 2 minutes until blood clot occurred. Two groups of contrast media were identified: (1) iohexol and iopamidol, which increased the clotting time, and (2) ioxaglate, diatrizoate, and ioxitalamate, for which all clotting times were over 30 minutes and no FpA generation occurred.

In vitro interactions of gadolinium DOTA meglumine and gadolinium DTPA meglumine on hemostasis.

The interactions of two gadolinium complexes (Gd-DOTA meglumine and Gd-DTPA meglumine) with hemostatic function have been analyzed using: (1) coagulation reactions (extrinsic and intrinsic pathways and fibrinoformation) and (2) platelet function investigations (aggregation, release of Ca++ and ATP after stimulation with collagen 2.5 micrograms/mL). The data obtained with Gd-DTPA meglumine (Mgl) exhibited a significant increase of the intrinsic coagulation pathway and a delay in fibrinoformation, although there is no alteration of the effect of thrombin on fibrinogen (fibrinopeptid A determinations). Platelet aggregation and release are moderately modified. In contrast, Gd-DOTA Mgl exerts no effect on the coagulation system and only minor effects on platelet functions. It is suggested that at least one mechanism involves the complexation of ionized calcium because Gd-DTPA Mgl and Gd-DOTA Mgl complex, respectively, 45% and 23% of ionized calcium in plasma. However, other mechanisms such as an alteration of fibrin polymerization are not unlikely.

Haemocompatibility and biological course of carbonaceous composites for cardiovascular devices.

A new class of carbonaceous composites has been developed for cardiovascular devices. The aim of the present study, performed in dogs, was to test the immediate blood compatibility of these materials when inserted within the vascular bed. Biocompatibility studies were performed on vascular cylinders (6 mm i.d.) and intra-atrial implants. The specimens were examined sequentially by SEM at 10, 20, 30, 180 s and 10 min after re-establishment of the blood flow. Patency of the vascular cylinders was tested during the second and third postoperative month by Doppler ultrasound investigations; specimens were examined by light and electron microscopy (scanning and transmission) at 15, 60 and 110 d following implantation. As early as 10 s after re-establishment of the blood flow platelet adhesion and a limited fibrin mesh with few erythrocytes developed on the material. Platelet aggregates were only observed on intravenous implants. Except in the case of the intravenous insert, no thrombosis developed at the contact of intra-arterial or intracardiac implants. After 15 d it was completely covered by a fibrocellular layer (3-5 cells thick) consisting of large myofibroblasts with microfilaments, newly synthesized collagen and elastin. Endothelial-like cells developed and were completed 2 mnth after implantation. However, deposits present inside and outside the fibrocytic cells of the newly developed tissue were observed corresponding to carbon peaks as indicated by wavelength dispersive X-ray microanalysis.

Mechanical properties and biocompatibility of two polyepoxy matrices: DGEBA-DDM and DGEBA-IPD.

The aim of this paper was to study the biocompatibility and mechanical properties of materials for orthopaedic and odontologic surgical use. The products used were obtained by polycondensation of a diepoxy resin (DGEBA) with two curing agents (DDM or IPD). The materials present a slight swelling in liquid medium and their thermomechanical properties are hardly affected after 12 month implantation. The absence of molecular desorption in isotonic liquid and human serum confirms their hydrolytic stability and thus their inertia. These materials do not give rise to an intolerance reaction by neighbouring tissues during implantation time (1 d to 12 month).

In vitro biocompatibility evaluation of a heparinizable material (PUPA), based on polyurethane and poly(amido-amine) components.

The biocompatibility of a new heparinizable material based on polyurethane and poly(amido-amine) (PUPA) was evaluated both in the heparinized and non-heparinized forms. The quantity of heparin present on the material was measured using radiolabelled heparin and biological tests. Heparin release in plasma from heparinized PUPA was investigated using in vitro methods. The behaviour of PUPA towards cellular and plasmatic blood components was studied. The influence of sterilization on the cytocompatibility response of both heparinized and non-heparinized PUPA was investigated; gamma-rays were found to be a suitable method of sterilization as no toxic response was noticed.

Immunolocalization of factor V in adult and fetal rat liver.

Immunolocalization of the factor V was performed in adult and fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a mono-specific antiserum anti rat factor V. In all the cases (perfused and non-perfused adult liver and fetal one) the observation of the sections has revealed an intense circular or granular labelling into all the hepatocytes whatever was their localization in the hepatic lobule. Hepatic endothelial cells seemed to be negative for factor V and this aspect of our results was discussed.

In vitro study of the inhibition of coagulation induced by different radiocontrast molecules.

The anticoagulant activity of seven intravascular radiocontrast molecules (RCM) was evaluated in different in vitro systems using citrated human plasma. Each RCM was tested in a concentration range of 5 to 50 mM. The thrombin time and the reptilase time showed a dose-dependent lengthening of fibrinoformation, the recording of fibrinoformation exhibited a significant delay of fibrin monomer generation and polymerization although the amplitude of the fibrinoformation was not decreased. The interfering effect with fibrin clot formation impairs also global coagulation tests and monospecific coagulation tests using fibrinoformation as the final step of the assay, but a possible interaction between RCM and some specific coagulation factors cannot be excluded. RCM potentiated the anti-thrombin action of heparin but the inhibition or delay of fibrinoformation is not related to an antithrombinic effect of contrast media. The thrombin amidolytic activity is not modified by RCM but the generation of FpA is delayed and decreased. The ultrastructure of the fibrin clot is not altered at the end of the polymerization.