Victor Spicer

An Improved Model for Prediction of Retention Times of Tryptic Peptides in Ion Pair Reversed-phase HPLC Its Application to Protein Peptide Mapping by Off-Line HPLC-MALDI MS

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

Collisional damping interface for an electrospray ionization time-of-flight mass spectrometer

A new interface between atmosphere and high vacuum has been developed for orthogonal injection of electrosprayed ions into a time-of-flight mass spectrometer. A small rf quadrupole operating at 100 mtorr (1.33 × 10−4 bar) is its key element. Ions enter the quadrupole with velocities acquired in the free expansion/declustering process. As they pass through the quadrupole their motion is constrained by the rf field. Meanwhile, they lose energy by collisions with the gas molecules. The time delays of ions passing through the quadrupole have been measured in order to determine the average velocities of the ions and the factors determining this value. In addition, a simple computational model based on a Monte Carlo approach has been developed to simulate the ion motion; it shows a considerable decrease in both transverse and axial ion velocity components. As the result of collisional damping the interface provides a dramatic improvement in the overall quality of the ion beam transported into the mass spectrometer. Both resolution and sensitivity of the time-of-flight instrument are improved and mass-to-charge ratio discrimination is greatly reduced.

ORTHOGONAL INJECTION OF MATRIX-ASSISTED LASER DESORPTION/IONIZATION IONS INTO A TIME-OF-FLIGHT SPECTROMETER THROUGH A COLLISIONAL DAMPING INTERFACE

Ions are produced from a conventional matrix-assisted laser desorption/ionization (MALDI) target by irradiation with a nitrogen laser pulsed at 20 Hz. After being cooled by collisions in an RF-quadrupole ion guide, the ions enter an orthogonal-injection TOF mass spectrometer, already used for electrospray. The collisional cooling spreads the ions out along the axis of the quadrupole to produce a quasi-continuous beam, which is then pulsed into the mass spectrometer at a repetition rate of about 4 kHz. Approximately five ions enter the mass spectrometer with each injection pulse, and these are detected using single-ion counting and registered in a TDC with 0.5 ns resolution. The performance of the instrument is similar to that obtained with an ESI source. A uniform mass resolution of about 5000 (full width at half maximum definition) is routinely obtained for molecular weights up to about 6000 Da, with mass accuracy around 30 ppm. The sensitivity for peptides is in the low femtomole range. The mass range is currently limited by the low energy (5 keV) of the ions at the detector, although ions of cytochrome C (12 359 Da) have been detected. The performance of the instrument for peptides is competitive with delayed-extraction MALDI in the usual axial geometry, but with the advantage of mass-independent focusing conditions, and a simple two-point calibration procedure. However, the most important advantages result from the nearly complete decoupling of the ion production from the mass measurement. In the usual MALDI experiment the instrument must be carefully adjusted for optimum performance, and the optimum parameters depend on the matrix and the method of sample preparation. As a result of the decoupling, the performance of the instrument is independent of source conditions. This allows much greater flexibility to experiment with different matrices, different substrates (including insulating substrates), and different laser wavelengths, pulse widths and fluences. Because of the decoupling, the design also allows convenient use of both ESI and MALDI sources (and possibly others) on the same spectrometer.

Citrus Chlorophyllase Dynamics at Ethylene-Induced Fruit Color-Break: A Study of Chlorophyllase Expression, Posttranslational Processing Kinetics, and in Situ Intracellular Localization

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.

Novel Linac II Electrode Geometry for Creating An Axial Field in a Multipole Ion Guide

Tandem instruments, such as triple-quadrupole or quadrupole-time-of-flight mass spectrometers, often use collisional damping ion guides for the purpose of cooling and focusing the primary ion beam, or as a collision cell for tandem mass spectrometry experiments. A small axial field to reduce the ion residence time in such devices can give considerable improvements in performance. Reduction of the residence time reduces adduct formation caused by unwanted gas-phase reactions and allows multiple reaction monitoring (MRM) and some other types of scan to be carried out more rapidly without the risk of reaction cross-talk. Here we propose a novel electrode arrangement for creation of a suitable axial field in a multipole ion guide without significantly reducing the m/z window. As an example, a second set of four electrodes is added to an existing quadrupole collision cell. The same DC potential is applied to all four extra electrodes, which are shaped in the longitudinal direction to create a suitable axial field inside the device. The potential of these electrodes and the DC bias of the main rods determine the axial field strength. The idea was tested on a modified quadrupole collision cell of a MALDI-QqTOF instrument and experimental results are presented here, as well as a simplified theory of operation.

Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

The effect of laser profile, fluence, and spot size on sensitivity in orthogonal‐injection matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The influence of incident laser parameters on sensitivity in matrix-assisted laser desorption/ionization (MALDI) has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N(2) laser and a Nd:YAG laser using 2-m long optical fibers. The N(2) laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N(2) laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. Thus, the optimum sensitivity is achieved at high fluence. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. The results have particular relevance for imaging MALDI, where sensitivity and spatial resolution are important figures of merit.

Quantitative proteomic analysis of the cellulolytic system of Clostridium termitidis CT1112 reveals distinct protein expression profiles upon growth on α-cellulose and cellobiose

UNLABELLED Clostridium termitidis CT1112 is an anaerobic, mesophilic, cellulolytic bacterium with potential applications in consolidated bioprocessing of lignocellulosic biomass. To understand how C. termitidis degrades lignocellulose, iTRAQ-based 2D HPLC-MS/MS proteomics was used to measure protein expression in cell lysates and extracellular (secretome) fractions of C. termitidis grown on α-cellulose and cellobiose at both exponential and stationary growth phases. Exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), hemicellulases including xylanases (GH8, GH10, GH11, GH30) and mannanase (GH26) as well as extracellular adhesion proteins and cellulosome associated proteins, exhibited higher expression on cellulose-grown cells. The expression of these proteins increased with a decrease in growth rate. Non-cellulosomal proteins however did not change significantly between substrate conditions, although there were a few exceptions. Collectively, these would contribute to hydrolysis of lignocellulosic material for uptake through ABC sugar transport proteins. On cellobiose, chitinases (GH18) were expressed abundantly. Although a large number of proteins were shared between the fractions analyzed, some proteins were detected exclusively in the cellular fraction, while others were detected in the secretome. This study reports for the first time on the cellulolytic machinery employed by C. termitidis to hydrolyze cellulosic substrate and provides an understanding of how this microbe deconstructs biomass. BIOLOGICAL SIGNIFICANCE The genome of C. termitidis CT1112 contains genes for a wide variety of carbohydrate active enzymes. Based on bioinformatics analyses, many of these genes appear to encode cellulosome-associated proteins, while others may be secreted extracellularly. To understand how C. termitidis degrades and depolymerizes cellulosic substrates, cells were grown on simple and complex carbohydrates, and quantitative 4-plex iTRAQ-based 2D HPLC-MS/MS proteomics was applied to measure protein expression levels in biological replicates of both cell lysates and extracellular protein (secretome) fractions, at exponential and stationary phases of growth. The resulting data have provided insight into the range of substrates that may be hydrolyzed by C. termitidis, and may be useful in determining potential industrial applications of C. termitidis in biomass to bioenergy production via consolidated bioprocessing.

Proteomics-based metabolic modeling and characterization of the cellulolytic bacterium Thermobifida fusca

BackgroundThermobifida fusca is a cellulolytic bacterium with potential to be used as a platform organism for sustainable industrial production of biofuels, pharmaceutical ingredients and other bioprocesses due to its capability of potential to convert plant biomass to value-added chemicals. To best develop T. fusca as a bioprocess organism, it is important to understand its native cellular processes. In the current study, we characterize the metabolic network of T. fusca through reconstruction of a genome-scale metabolic model and proteomics data. The overall goal of this study was to use multiple metabolic models generated by different methods and comparison to experimental data to gain a high-confidence understanding of the T. fusca metabolic network.ResultsWe report the generation of three versions of a metabolic model of Thermobifida fusca sp. XY developed using three different approaches (automated, semi-automated, and proteomics-derived). The model closest to in vivo growth was the proteomics-derived model that consists of 975 reactions involving 1382 metabolites and account for 316 EC numbers (296 genes). The model was optimized for biomass production with the optimal flux of 0.48 doublings per hour when grown on cellobiose with a substrate uptake rate of 0.25 mmole/h. In vivo activity of the DXP pathway for terpenoid biosynthesis was also confirmed using real-time PCR.Conclusionsi Tfu296 provides a platform to understand and explore the metabolic capabilities of the actinomycete T. fusca for the potential use in bioprocess industries for the production of biofuel and pharmaceutical ingredients. By comparing different model reconstruction methods, the use of high-throughput proteomics data as a starting point proved to be the most accurate to in vivo growth.

Identification of Variants of the High Plains virus Infecting Wheat in Kansas

The properties of two virus isolates (U04-82 and U04-83) obtained from two wheat (Triticum aestivum) plants expressing mosaic symptoms were investigated using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), time-of-flight mass spectrometry (TOFMS), and infection of wheat with resistance to Wheat streak mosaic virus (WSMV). The coat protein mass was estimated by SDS-PAGE as approximately 32 kDa for U04-82 and 30 kDa for U04-83. The amino acid sequence of the coat protein of U04-82 was 99.6 and 85.5% identical to two isolates, ABC58222 and TX96, respectively, of High Plains virus (HPV) described from Texas. U04-82 was transmitted by wheat curl mites and caused significant yield reductions in wheat resistant to WSMV. U04-83 was actually two distinct virus isolates whose capsid protein amino acid sequences were only 57 and 50% similar to that of TX96. Antiserum prepared to a synthetic peptide from the sequence of the U04-83 isolate recognized the two U04-83 isolates, but not the U04-82 isolate.