Passive Elution Research Articles

Gel Protein Extraction's Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.

Comparison of simple and rapid extracting methods of free-tags Mycobacterium tuberculosis protein 64 Recombinant Protein from polyacrylamide gel: Electroelution and the optimized passive elution.

ABSTRACTIn this study, the Mycobacterium tuberculosis protein 64 (MPT64) protein was constructed without any tags to facilitate the purification using column affinity chromatography, but the MPT64 must be obtained as a pure protein. This study was purpose to ensure the efficient extracting method to purify protein MPT64 directly from the polyacrylamide gel. The crude extract of extracellular protein containing MPT64 protein was separated into single protein band and the targeted protein which is located in the size of 24 kDa was excised. Each of the six bands was collected in a sterile microtube to be eluted using electroelution and the optimized of the passive-elution method. Both the elution methods demonstrated the purity level of the MPT64 protein by detecting a solely band on the gel at the 24 kDa. Among the variety of passive-elution time, the highest MPT64 protein concentration was 0.549 mg/ml after elution for 72 h. However, the electroelution result provided higher MPT64 protein concentration, i.e., 0.683 mg/mL. However, based on the recognition of the purified MPT64 protein on commercial detection kit of MPT64 protein, it showed that the positive result was only showed by the passive-elution extracting protein. Therefore, for purifying the protein MPT64 from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, the efficient method was passive elution.

The Use of a Novel Antimicrobial Implant Coating In Vivo to Prevent Spinal Implant Infection.

A controlled, interventional animal study. Spinal implant infection (SII) is a devastating complication. The objective of this study was to evaluate the efficacy of a novel implant coating that has both a passive antibiotic elution and an active-release mechanism triggered in the presence of bacteria, using an in vivo mouse model of SII. Current methods to minimize the frequency of SII include local antibiotic therapy (vancomycin powder), betadine irrigation, silver nanoparticles, and passive release from antibiotic-loaded poly(methyl methacrylate) cement beads, all of which have notable weaknesses. A novel implant coating has been developed to address some of these limitations but has not been tested in the environment of a SII. A biodegradable coating using branched poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS) polymer was designed to deliver antibiotics. The in vivo performance of this coating was tested in the delivery of either vancomycin or tigecycline in a previously established mouse model of SII. Noninvasive bioluminescence imaging was used to quantify the bacterial burden, and implant sonication was used to determine bacterial colony-forming units (CFUs) from the implant and surrounding bone and soft tissue. The PEG-PPS-vancomycin coating significantly lowered the infection burden from postoperative day 3 onwards (P < 0.05), whereas PEG-PPS-tigecycline only decreased the infection on postoperative day 5 to 10 (P < 0.05). CFUs were lower on PEG-PPS-vancomycin pins than PEG-PPS-tigecycline and PEG-PPS pins alone on both the implants (2.4 × 10, 8.5 × 10, and 1.0 × 10 CFUs, respectively) and surrounding bone and soft tissue (1.3 × 10, 4.8 × 10, and 5.4 × 10 CFUs, respectively) (P < 0.05). The biodegradable PEG-PPS coating demonstrates promise in decreasing bacterial burden and preventing SII. The vancomycin coating outperformed the tigecycline coating in this model compared to prior work in arthroplasty models, highlighting the uniqueness of the paraspinal infection microenvironment. N/A.

Comparison of electrode impedance measures between a dexamethasone-eluting and standard Cochlear™ Contour Advance® electrode in adult cochlear implant recipients

ObjectiveTo compare the difference in electrode impedance across discrete time points to 24 months post-activation for two groups of adult cochlear implant recipients, one using an investigational perimodiolar (Contour Advance®) array augmented with 40% concentration weight per weight (w/w) dexamethasone (the Drug Eluting Electrode, ‘DEE’ Group), and the other the commercially available Contour Advance (’Control’ Group). DesignTen adult subjects were implanted with the DEE and fourteen with the Control. Electrode impedances were measured intra-operatively, one-week post-surgery, at initial activation (approximately two-weeks post-surgery), and at approximately one, three, six, 12 and 24 months post-activation. Two different impedance measurements were obtained: 1) in MP1+2 mode using Custom Sound programming software; and 2) 4-point impedance measures utilising BP+2 stimulation mode with recording on non-stimulating electrodes. Data were analysed with respect to both impedance averaged across all electrodes, and impedance for electrodes grouped into basal, middle and apical sections. ResultsGroup mean MP1+2 impedance for the DEE was significantly lower than for the Control at all post-operative time points examined, and for each of the basal, middle and apical cochlear regions. Group mean 4-point impedance was significantly lower for the DEE than the Control in the basal region at six, 12 and 24 months post-activation and in the middle region at 12- and 24-months post-activation. The pattern of change in MP1+2 impedance differed significantly in the early post-operative period prior to device activation. A significant 4.8 kOhm reduction in impedance between surgery and one-week was observed for the DEE group but not for the Control. A 2.0 kOhm increase between the one and two week post-operative time points was observed for the Control but not for the DEE group. ConclusionWhile rates of adoption of different surgical approaches differed between the groups and this may have had a confounding effect, the results suggest that passive elution of dexamethasone from the investigational device was associated with a change in the intracochlear environment following surgical implantation of the electrode array, as evidenced by the lower electrode impedance measures.

059 Local anaesthesia eluting property of Coloplast Titan penile prosthesis hydrophilic coating: An in-vitro drug elution profile and a randomised double-blind clinical outcome study

Local anaesthesia eluting property of Coloplast Titan penile prosthesis hydrophilic coating: An in-vitro drug elution profile and a randomised double-blind clinical outcome study. A two-part study to provide proof-of-concept with in-vitro analysis of drug elution property, and to evaluate the clinical outcome with Titan penile prosthesis hydrophilic coating dipped in local anaesthesia agent. An in-vitro analysis of local anaesthesia eluting property of hydrophilic coating to determine the amount and duration of local anaesthesia elution, and examine if the addition of local anaesthesia agent will dilute anti-microbial property of hydrophilic coating. Following internal ethics approval, a pilot study was conducted with 40 men randomised to receive Titan’s penile prosthesis dipped with or without 0.75% Ropivacaine and 0.5% Marcaine. An independent third-party survey with objective measurement of pain score (visual analog scale, VAS), analgesia requirement (opioid dose equivalence) and time to penile prosthesis recycling. In vitro-release kinetics confirmed passive drug elution above the minimum inhibitory concentration. The minimum local anaesthetic concentration (MLAC) of 0.75% Ropivacaine and 0.5% Marcaine was sustained over the 14 days of in-vitro drug elution test. The hydrophilic coating with combination local anesthesia and antimicrobial drugs showed sustained elution and zone of microbial inhibition at day 1, 3, 7 and 14 (p<0.05). In the clinical study, patients with Titan penile prosthesis dipped in local anaesthesia reported lower VAS score (p<0.05), less analgesia requirement (p<0.05) and shorter time to penile prosthesis recycling (p=0.08).

Elution of Artificial Sputum from Swab by Rotating Magnetic Field-Induced Mechanical Impingement

Cotton-tipped applicator swabs are used as a collection device for many biological samples and its complete elution is a desired step for clinical and forensic diagnostics. Swabs are used to collect infectious body fluids, where the concentration of pathogens can range from 1 × 104 CFU/mL (colony forming units/mL) in respiratory-tract infections and 1 × 105 in urinary-tract infections, to up to 1 × 109 CFU/mL in salivary samples. These samples are then eluted and lysed, prior to DNA (De-oxy Ribonucleic Acid) analysis. The recovery of micro-organisms from a matrix of swab fibres depends on the nature of the body fluid, the type of the swab fibres, and the process of elution. Various methods to elute samples from swab include chemical digestion of fibres (~20% recovery), centrifugation (~58% recovery), piezoelectric vibration, or pressurized fluid-flow (~60% recovery). This study reports a magnetically-actuated physical impingement method for elution and recovery of artificial sputum samples from cotton fibres. A device has been fabricated to induce a rotating magnetic field on smaller magnetic particles in a vial that strikes the swab within a confined gap. Elution from the swab in this device was characterized using 2% Methyl cellulose in deionised water, loaded with fluorescent-tagged polystyrene beads and E. coli at various concentrations. The recovery efficiency was found to increase with both rotational speed and elution time, but plateaus after 400 RPM (Revolutions per minute) and 120 s, respectively. At a higher concentration of polystyrene beads (5 × 108 particles/mL), a maximum recovery of ~85% was achieved. With lower concentration, (1 × 105 particles/mL) the maximum efficiency (~92.8%) was found to be almost twice of passive elution (46.7%). In the case of E. coli, the corresponding recovery efficiency at 3.35 × 105 CFU/mL is 90.4% at 500 RPM and 120 s. This elution method is expected to have a wide applicability in clinical diagnostics.

In Vivo Efficacy of a “Smart” Antimicrobial Implant Coating

Postoperative infection is a devastating complication following arthroplasty. The goals of this study were to introduce a "smart" implant coating that combines passive elution of antibiotic with an active-release mechanism that "targets" bacteria, and to use an established in vivo mouse model of post-arthroplasty infection to longitudinally evaluate the efficacy of this polymer implant coating in decreasing bacterial burden. A novel, biodegradable coating using branched poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS) polymer was designed to deliver antibiotics both passively and actively. In vitro-release kinetics were studied using high-performance liquid chromatography (HPLC) quantification in conditions representing both the physiologic environment and the more oxidative, hyperinflammatory environment of periprosthetic infection. The in vivo efficacy of the PEG-PPS coating delivering vancomycin and tigecycline was tested using an established mouse model of post-arthroplasty infection. Noninvasive bioluminescence imaging was used to quantify the bacterial burden; radiography, to assess osseointegration and bone resorption; and implant sonication, for colony counts. In vitro-release kinetics confirmed passive elution above the minimum inhibitory concentration (MIC). A rapid release of antibiotic was noted when challenged with an oxidative environment (p < 0.05), confirming a "smart" active-release mechanism. The PEG-PPS coating with tigecycline significantly lowered the infection burden on all days, whereas PEG-PPS-vancomycin decreased infection on postoperative day (POD) 1, 3, 5, and 7 (p < 0.05). A mean of 0, 9, and 2.6 × 10(2) colony-forming units (CFUs) grew on culture from the implants treated with tigecycline, vancomycin, and PEG-PPS alone, respectively, and a mean of 1.2 × 10(2), 4.3 × 10(3), and 5.9 × 10(4) CFUs, respectively, on culture of the surrounding tissue (p < 0.05). The PEG-PPS coating provides a promising approach to preventing periprosthetic infection. This polymer is novel in that it combines both passive and active antibiotic-release mechanisms. The tigecycline-based coating outperformed the vancomycin-based coating in this study. PEG-PPS polymer provides a controlled, "smart" local delivery of antibiotics that could be used to prevent postoperative implant-related infections.

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA

ABSTRACTThe PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.

OFFGEL fractionation of peptides: Where really is your sample?

Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.

Red blood cells release factors with growth and survival bioactivities for normal and leukemic T cells

Human red blood cells are emerging as a cell type capable to regulate biological processes of neighboring cells. Hereby, we show that human red blood cell conditioned media contains bioactive factors that favor proliferation of normal activated T cells and leukemic Jurkat T cells, and therefore called erythrocyte-derived growth and survival factors. Flow cytometry and electron microscopy in parallel with bioactivity assays revealed that the erythrocyte factors are present in the vesicle-free supernatant, which contains up to 20 different proteins. The erythrocyte factors are thermosensitive and do not contain lipids. Native polyacrylamide gel electrophoresis followed by passive elution and mass spectrometry identification reduced the potential erythrocyte factors to hemoglobin and peroxiredoxin II. Two-dimensional differential gel electrophoresis of the erythrocyte factors revealed the presence of multiple hemoglobin oxy-deoxy states and peroxiredoxin II isoforms differing in their isoelectric point akin to the presence of β-globin chains. Our results show that red blood cells release protein factors with the capacity to sustain T-cell growth and survival. These factors may have an unforeseen role in sustaining malignant cell growth and survival in vivo.

A Novel Chip-based Electroelution System for Rapid and Efficient Recovery of Intact Proteins from Polyacylamide Gels

A Novel Chip-based Electroelution System for Rapid and Efficient Recovery of Intact Proteins from Polyacylamide Gels

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

Increased antiviral activity of microscale‐purified HuIFNα8 (human interferon α8) over HuIFNα2b in Hep‐2 cells challenged with Mengo virus

Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.

SDS-PAGE Focusing: Preparative Aspects

As a followup of our previous report (Zilberstein, G.; Korol, L.; Antonioli, P.; Righetti, P. G.; Bukshpan, S. Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a novel method is here reported for small-scale prefractionation of complex protein mixtures, for subsequent proteome analysis, based on mass separation of SDS-protein micelles not in a gel matrix, but in liquid cationic polymers assembled in a multicompartment electrolyzer (MCE) in a stepwise fashion at discrete and increasing levels of positive charges (from 3 to 28 mM), the neighboring chambers being separated by neutral agarose membranes. Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method of SDS-PAGE focusing exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path and trapped into different compartments of the MCE device via interaction (and subsequent charge neutralization) with cationic polymers of fixed (but increasing from chamber to chamber from cathode to anode) charge density. Minimization of migration of the liquid cationic polymers is obtained via use of low voltage and by arranging for a buffer conductivity gradient along the migration path. The present setup has the advantage of high protein recoveries (up to 90%) without any contamination from ungrafted monomers and catalysts, as occurring in proteins recovered by passive elution from gel matrixes. Additionally, resolution can be fine-tuned by selecting cationic polymers of varying charge density in microstep increments. The cationic polymers, of desired charge density and proper viscosity, are prepared by standard polymerization conditions, can be easily precipitated and washed free of monomeric contaminants, and stored in a dry form for subsequent use.

Evaluation of the SELDI‐TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate

The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.

Isolation of smooth‐type lipopolysaccharides to electrophoretic homogeneity

The high structural heterogeneity of smooth-type lipopolysaccharides (LPS) enormously complicates the isolation of their constituent molecular species. Proof of concept is given here on the feasibility of using preparative slab-PAGE to isolate highly homogeneous smooth-type LPS glycoforms. LPS species (from 3.6 to 14.2 kDa) from Escherichia coli K-235 were separated by preparative slab-PAGE and recovered by utilizing the combined on-gel LPS reverse staining, extrusion, and passive elution techniques. As a result, 15 electrophoretically pure LPS fractions were obtained. The LPS content in the recovered fractions ranged from 280 ng (intermediate mobility glycoforms) to 411 mug (highest mobility glycoforms). The quantities of LPS fractions were sufficient to allow quantitation of the Limulus amebocyte lysate (LAL) activities of these distinct-molecular-mass LPS species, in the range from (1.1 +/- 0.1)x10(3) to (8.7 +/- 0.3)x10(5) endotoxin units (EU)/mL, by standard LAL assay. We have thus definitively demonstrated that slab-PAGE may be a suitable platform to more selectively purify individual glycoform fractions from smooth-type LPS.

The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

BackgroundThe aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM).Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS).ResultsMass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM.ConclusionProtocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed.

Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK.

We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high-efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab-gel separation and electrospray ionization-mass spectrometry (ESI-MS) of LPS-derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O-deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse-stained LPS bands using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The quantities (30-40 microg) obtained from the two gel-resolved LPS bands were sufficient to allow MALDI-TOF-MS detection of O-deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high-molecular-weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low-molecular-weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI-TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal-to-noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI-TOF-MS led to a higher sensitivity of detection of less abundant low-molecular-weight LPS glycoforms. Taken together, this and our previous study on gel-micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel-separated LPS band, their relative abundance and oligosaccharide sequences.