Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS

Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm×150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC-MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.     

Counting total cell numbers microscopically on stained films

Two main sources of error in the conventional stained film cell count method, i.e. errors from imperfect spreading of the suspension and uneven distribution of cells in the film, have been eliminated by counting all the cells in the film. Staining solution and sodium alginate were added to the suspension before spreading over the slide. With a microsyringe, 20-μl suspension was spread in two bands of ca 1.7 mm × 60 mm each. Then total cells could be counted easily in the x 100 magnification field by moving the slide in one direction. Results were satisfactory. Coefficients of variation were 2.6 and 1.1% in two separate yeast determinations each with five replicates. For counting higher magnifications, a suitable image analyser for automating the method is believed worthy of study.     

Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III-I-IV for PpcB, as opposed to I-IV-III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.     

Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain

Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxylapatite chromatography in 8 M-urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low-ionic-strength buffer. Hence, they can provide a source for further biochemical studies.     

Sequential extractions: A new way for protein quantification—data from peanut allergens

Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by the Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of them in solution. Extracting all of the contents is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein contents and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If all proteins can be extracted with sufficient extraction times, the protein contents in the peanut matrix can be calculated using a mathematical summation formula. This sum should be all proteins in the matrix. The five-step sequential extraction method can provide a means to quantify certain proteins in the matrix.     

Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III-I-IV for PpcB, as opposed to I-IV-III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of tetrahydropalmatine enantiomers in dogs

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.     

The electrostatic contribution to interactions of some enzymes with polyelectrolytes

To explain the inhibitory action of polyelectrolytes on enzymes and, in particular, to define potentially reactive zones for the binding of polyelectrolyte, the electric potential of enzymes lactate dehydrogenase and glutamate dehydrogenase was calculated using the solution of the Poisson-Boltzmann equation by a numerical method with the use of the Gauss-Seidel relaxation method at three pH values: 6.5, 7.0, and 8.0 and three values of ionic strength: 50, 100, and 150 mm. On the basis of these calculations and their visualization, representative sites for favorable binding of polyanions were determined as extended areas on the surface of proteins with the positive potential in the neutral pH region. It was shown that there is a correlation between the area of positive potential and the efficiency of enzyme inactivation for a number of pH values and concentrations of salt for two enzymes. The calculations performed allowed one to explain the inhibitory action of polyelectrolytes on the specified enzymes to understand the difference between the values of polyelectrolyte inactivation constants for the two enzymes and estimate the minimal areas of the positive potential on the protein surface that provide their effective inhibition.     

Genetically engineered binding proteins as biosensors for fermentation and cell culture

The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both proteins respond to their ligands in the micromolar range. The proteins can be stored at 4 degrees C for at least 5 months. Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges. The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours. Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes. The potential applications of GBP were studied in yeast fermentation and E. coli fermentations in three different scales: 150 mL, 5 mL, and 100 microL. The results were compared with an YSI 2700 Chemistry Analyzer. Although the latter could not give reliable results for the E. coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation. Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 microL) and the results were also compared with those obtained with YSI. Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 microL) prevented it from being used in the 100-microL cell culture. Because of their small sample volume requirements (less than 1 microL) and high sensitivity, the assays described here might find wide applications in high-throughput bioprocessing.     

Growth of Strombus gigas (Gastropoda: Strombidae) snail in 4 environments of Quintana Roo, Mexico]

The growth rate of queen conch cultured in pens was studied from October 1993 to March 1994. Sixteen pens (50 m2 each, four pens per environment), were set in four environments: Thalassia, Thalassia-sand, Sand and Coral within a reef lagoon on Punta Gavilan and Banco Chinchorro. Twenty conchs were introduced in each pen (sizes: 100-120, 120-140, 140-160 and 160-180 mm shell length) and measured monthly to the nearest mm. Growth rate was assessed by two methods: a) shell marginal mean increase and b) the Gulland-Holt method considering all conch within pens. In the first method, the environment Sand had the highest growth (3.21 +/- 0.26 mm/month) at Punta Gavilan, whereas at Banco Chinchorro, highest growth was recorded in Coral (2.31 +/- 0.44 mm/month). Considering the second method, highest asymptotic length conch in Punta Gavilan occurred in Thalassia-sand (287.5 mm), whereas in Banco Chinchorro the highest asymptotic length was measured in Sand (318.1 mm). There were significant differences in growth between sites; juvenile growth is related with habitat quality mainly food availability.     

Telomerase catalyzed fluorescent probes for sensitive protein profiling based on one-dimensional microfluidic beads array

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360-480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N=3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site "sandwich" immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per mul sample can be determined specifically. The expression changes of p53, c-myc and beta-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.     

One-dimensional proteomic mapping of human liver cytochromes P450

A method for constructing one-dimensional proteomic maps (1D-PM) based on mass spectrometric identification of proteins from adjacent slices of one-dimensional electrophoregram has been developed. For the proteomic mapping, gel lanes were sectioned into slices less than 0.2 mm thick and each slice was subjected to enzymatic hydrolysis. The resultant mixture of peptide fragments was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteins were identified by the mass spectra obtained. Data on peptide fragments and corresponding identified proteins were presented as a 1D-PM. Proteomic maps were constructed by assigning individual proteins to gel slices based on number of matching peptides in a corresponding MS-data. On 1D-PM of human liver microsomal fraction, 18 proteins were identified in the region of 40–65 kDa. These included 12 membrane proteins belonging to the superfamily of cytochromes P450. Pooling of mass spectrometric data, obtained from several adjacent gel slices (molecular zooming) increased sequence coverage of CYP2A (cytochrome P450 family 2A). The maximal coverage of 66% significantly exceeded the level of 48% that could be obtained using one (even the most informative) slice. This method can be applied to the proteomic profiling of membrane-bound proteins.     

Two methods for proteomic analysis of formalin‐fixed,paraffin embedded tissue result in differential protein identification,data quality,and cost

Formalin‐fixed paraffin‐embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC‐MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in‐solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre‐analytical variations and analyzed with three technical replicates by LC‐MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre‐analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.     

Conductance catheter measurements of lumen area of stenotic coronary arteries: theory and experiment

An injection of saline solution is required for the measurement of vessel lumen area using a conductance catheter. The injection of room temperature saline to displace blood in a vessel inevitably involves mass and heat transport and electric field conductance. The objective of the present study is to understand the accuracy of conductance method based on the phenomena associated with the saline injection into a stenotic blood vessel. Computational fluid dynamics were performed to simulate flow and its relation to transport and electric field in a stenotic artery for two different sized conductance catheters (0.9 and 0.35 mm diameter) over a range of occlusions [56-84% cross-sectional area (CSA) stenosis]. The results suggest that the performance of conductance catheter is dependent on catheter size and severity of stenosis more significantly for 0.9 mm than for 0.35 mm catheter. Specifically, the time of detection of 95% of injected saline solution at the detection electrodes was shown to range from 0.67 to 3.7 s and 0.82 to 0.94 s for 0.9 mm and 0.35 mm catheter, respectively. The results also suggest that the detection electrodes of conductance catheter should be placed outside of flow recirculation region distal to the stenosis to minimize the detection time. Finally, the simulations show that the accuracy in distal CSA measurements, however, is not significantly altered by whether the position of detection electrodes is inside or outside of recirculation zone (error was within 12% regardless of detection electrodes position). The results were experimentally validated for one lesion geometry and the simulation results are within 8% of actual measurements. The simulation of conductance catheter injection method may lead to further optimization of device and method for accurate sizing of diseased coronary arteries, which has clinical relevance to percutaneous intervention.     

Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

Purification of the Specific Plasma Membrane Proteins from the Surface of Sperm Cells of Zea mays

Pollen samples of 6 varieties of Zea mays L. were used to isolate the viable sperm cells. After being probed with N-hydroxysuccinimido-biotin (NHS-biotin), the sperm cell plasma membrane proteins were compared with each other using the method of Western blotting. Results showed that there was no significant difference among varieties. The molecular weights of probed plasma membrane proteins were concentrated on 91,60,43,30 and 17 kD. Immunochemical method was adopted for further purification of sperm plasma membrane protein preparation which was some- what contaminated with cell organelles. After the cell organelles were isolated from etiolated seedlings of Zea mays by sucrose density gradient super centrifugation, the crude membrane proteins of organelles, endoplasm reticulum, mitochondria, Golgi body and plasmolemma were respectively used as antigen to immunize Guinea pig. The antibody was obtained from respective antiserum, then further used to produce immuno-affinity absorbent. After the solution of membrane proteins of sperm cells passed through the column, some proteins probed whth NHS-biotin were identified. Two major proteins probed with NHS-biotin were considered to be sperm cell specific. The size of these proteins in SDS-PAGE was about 65 kD, 22 kD, respectively.     

Protein variability in Meloidogyne spp. (Nematoda:Meloidogynidae) revealed by two-dimensional gel electrophoresis and mass spectrometry

Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.