MALDI-time Of Flight Research Articles

Comparison of serum protein profiles of Borrelia burgdorferi-positive Bernese Mountain Dogs and dogs of other breeds using MALDI-TOF technique.

The aim of the study was to compare the serum protein profile of Bernese Mountain Dogs (BMDs) reacting positive for Bb in snap testing with the serum protein profile of dogs of other breeds (healthy and with clinical borreliosis) using the MALDI time-of-flight (MALDI-TOF) technique. The observations included five groups of dogs. BMDs reacting positively to Bb in snap serological testing and showing symptoms of borreliosis (group 1), BMDs for which no borreliosis symptoms were determined but with seropositivity for Bb determined with snap serological tests (group 2), clinically healthy BMDs with no antibodies for Bb found in the serum (group 3), five dogs of different breeds, reacting positively in serological testing, in which borreliosis symptoms were observed (group 4), clinically healthy dogs of different breeds with negative reaction in tests towards Bb (group 5). A proteomic analysis demonstrated the presence of five identical protein fractions among all five groups. An additional two protein fractions of approximately 7.630 and 15.260 kDa were found in all the serum samples obtained from the dogs positive for borrelia in a snap test, both in those exhibiting symptoms of borreliosis, and seropositive BMDs not presenting symptoms of the disease. These two additional protein fractions may be used to differentiate between seropositive and seronegative B. burgdorferi dogs and may be considered a seropositivity marker, however, it cannot be used to differentiate between animals with the clinical form of the disease and those that are only seropositive.

Protein profiling analysis based on matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry and its application in typing Streptomyces isolates

Marine Streptomyces is a potential source of novel bioactive natural products in medicine and agriculture. The current discrimination and screening method of Streptomyces isolates is not accurate and time-consuming, and a novel method is necessary. In this study, a protein profiling method based on an ultrahigh resolution 15 T Matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) was established and applied for differentiation and bioactivity screening of marine Streptomyces isolates. To obtain robust protein profiling, the effects of the protein extraction method, the matrix-solvent, the sample deposition mode, and the culture time of isolates on protein profiling were thoroughly studied, the optimal conditions were obtained. To evaluate the performance of the developed MALDI-FTICR MS method, MALDI-time of flight (TOF) MS and 16S rRNA were applied in parallel to analyze 25 marine Streptomyces isolates. We found that the clustering result of MALDI-FTICR MS was more similar to that of 16S rRNA than MALDI-TOF MS. And MALDI-FTICR MS could effectively indicate the antibacterial activity of Streptomyces isolates against three plant pathogenic bacteria including Xanthomonas campestris, Xanthomonas oryzae and Erwinia carotovora. Furthermore, a differential protein/peptide was defined and successfully applied to predict antibacterial activity of blind samples. This study demonstrated that MALDI-FTICR MS has great potential to discriminate and screen complex microorganisms, especially those closely related strains.

Blood mass spectrometry Detects Residual Disease Better than Standard Techniques in Immunoglobulin light chain amyloidosis

In patients (pts) with AL amyloidosis, depth of hematologic response correlates with both organ response and overall survival. Our group has demonstrated that screening with a MALDI-time of flight (TOF) mass spectrometry (MS) is a quick, sensitive, and accurate means to diagnose and monitor the serum of pts with plasma cell disorders. Microflow liquid chromatography coupled with electrospray ionization and Q-TOF MS adds further sensitivity. Our goal was to assess the performance of MS in pts with AL who have been classified as complete response using conventional means.

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI).

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Imaging mass spectrometry of frontal white matter lipid changes in human alcoholics.

Chronic alcohol use disorders (AUD) are associated with white matter (WM) degeneration with altered myelin integrity. Matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) enables high throughput analysis of myelin lipid biochemical histopathology to help characterize disease mechanisms. This study utilized MALDI-IMS to investigatefrontal lobe WM myelin lipid abnormalities in AUD. Standardized cores of formalin-fixed WM from Brodmann Area 4 (BA4) and BA8/9 of 20 postmortem AUD and 19 control adult human brains were embedded in carboxymethyl-cellulose, cryo-sectioned (8μm), thaw-mounted onto indium tin oxide (ITO) -coated glass slides, and sublimed with 2,5-dihydroxybenzxoic acid (DHB) matrix. Lipids were imaged by MALDI-time of flight in the negative ionization mode. Data were visualized with FlexImaging software v4.0 and analyzed with ClinProTools v3.0. Principal component analysis (PCA) and data bar plots of MALDI-IMS data differentiated AUD from control WM. The dominant effect of AUD was to broadly reduce expression of sphingolipids (sulfatides and ceramides) and phospholipids. Data bar plots demonstrated overall similar responses to AUD in BA4 and BA8/9. However, differential regional effects of AUD on WM lipid profiles were manifested by non-overlapping expression or discordant responses to AUD for a subset of lipid ions. Human AUD is associated with substantial inhibition of frontal lobe WM lipid expression with regional variability in these effects. MALDI-IMS can be used to characterize the nature of AUD-associated lipid biochemical abnormalities for correlation with lifetime exposures and WM degeneration, altered gene expression, and responses to abstinence or treatment.

Chemical analysis of superconducting phase in K-doped picene

Potassium-doped picene (K3.0picene) with a superconducting transition temperature (TC) as high as 14 K at ambient pressure has been prepared using an annealing technique. The shielding fraction of this sample was 5.4% at 0 GPa. The TC showed a positive pressure-dependence and reached 19 K at 1.13 GPa. The shielding fraction also reached 18.5%. To investigate the chemical composition and the state of the picene skeleton in the superconducting sample, we used energy-dispersive x-ray (EDX) spectroscopy, MALDI-time-of-flight (MALDI-TOF) mass spectroscopy and x-ray diffraction (XRD). Both EDX and MALDI-TOF indicated no contamination with materials other than K-doped picene or K-doped picene fragments, and supported the preservation of the picene skeleton. However, it was also found that a magnetic K-doped picene sample consisted mainly of picene fragments or K-doped picene fragments. Thus, removal of the component contributing the magnetic quality to a superconducting sample should enhance the volume fraction.

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).Electrospray ionisation (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.Here we present an accessible approach for analysing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analysing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling). Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra. Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

In‐situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing

We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption-ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results.

Lipocalin-type prostaglandin D synthase protects against oxidative stress-induced neuronal cell death

L-PGDS [lipocalin-type PGD (prostaglandin D) synthase] is a dual-functional protein, acting as a PGD2-producing enzyme and a lipid transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. In the present study we demonstrate the protective effect of L-PGDS on H2O2-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H2O2-treated neuronal cells, and the L-PGDS level was highly associated with H2O2-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H2O2-mediated cell death. A cell viability assay revealed that L-PGDS protected against H2O2-induced cell death in a concentration-dependent manner. Furthermore, the titration of free thiols in H2O2-treated L-PGDS revealed that H2O2 reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight)-MS spectrum of H2O2-treated L-PGDS showed a 32 Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable with those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.

A new matrix of MALDI-TOF MS for the analysis of thiolate-protected gold clusters

Mass spectrometry (MS) can play an important role in determining the number of gold atoms per ligand molecule in thiolated Au clusters (CLs). However, the precise determination of the formulae of thiolated Au CLs is not a routine analysis and is still technically challenging. In this study, we report a new matrix for the matrix assisted laser desorption/ionization (MALDI) technique for thiolated Au CLs based on a commercially available α,β-diphenylfumaronitrile (DPF) matrix. This MALDI-time of flight (TOF) MS involving a DPF matrix allowed detection of intact Au25(SCH2CH2Ph)18 and Au25(SC12H25)18 ions with less fragmentation (i.e. softer ionization process) and stronger ion peak intensities compared to similar techniques containing a typical trans-2-[3-(4-tert-butylphenyl)-2-methyl-2propenylidene] malononitrile (DCTB) matrix for Au CLs. The DPF compound exhibited an absorbance maximum at about 336 nm, thereby making this matrix suitable for common N2 lasers (337 nm). The DPF matrix was suggested to function as an electron transfer agent from DPF−˙ to the Au CLs, leading to effective production of intact radical anions from Au CLs in negative ion operating mode.

Hydroxyapatite as a concentrating probe for phosphoproteomic analyses

A novel method for the selective enrichment of casein phosphoproteins/phosphopeptides (CPP) from complex mixtures is reported herein. This method employs ceramic hydroxyapatite (HA) as a solid-phase adsorbent to efficiently capture phosphoproteins and CPP from complex media. Casein was chosen as the model phosphoprotein to test the protocol. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. Casein fractions with different levels of phosphorylation were desorbed based upon the specific concentration of trifluoroacetic acid (TFA) included in the MALDI matrix. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried, and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach for the selection of casein/CPP resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The analytical process involved two steps requiring ∼2 h, excluding the time required for the enzymatic reaction. The alkaline phosphatase (AP)-assisted de-phosphorylation of tryptic CPP allowed the phosphorylation level of peptides to be calculated concurrently with MALDI-TOF MS and liquid chromatography-electrospray ionization–mass spectrometry (LC–ESI–MS/MS). The effectiveness of the extraction procedure assayed on eggshell phosphoproteins resulted in the identification of 5 phosphoproteins and 14 derived phosphopeptides with a phosphoprotein global recovery of ∼70% at least.

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.

Phosphorylated and non-phosphorylated forms of catechol O-methyltransferase in rat liver, brain and other tissues

Seven different forms of the enzyme COMT (catechol O-methyltransferase) were found in isolated rat hepatocytes by two-dimensional gel electrophoresis and immunoblotting: five small variants (S-COMT) and two large variants (L-COMT). The identities of these COMT forms were verified by tryptic fingerprinting using MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, and by amino acid sequencing using ESI-IT-MS/MS (electrospray ionization with ion-trap tandem MS). Analysis of tissue distributions showed that the S-COMT forms were highly expressed in liver and kidney, whereas L-COMT was expressed more strongly in other tissues. Both of the L-COMT forms were found in all of the tissues examined except the heart, which expressed only the most acidic form, and the kidney, which expressed only the most basic form. Subcellular fractionation revealed the presence of both S-COMT and L-COMT in soluble, as well as sedimentable, fractions, suggesting that they should be classified by size rather than (as previously) by localization. Several of the S-COMT forms were N-acetylated, and the two most acidic forms were found to be phosphorylated at Ser(260). One of the latter was unique to liver cells; the other was also found in kidney, brain and thymus. Among the non-phosphorylated S-COMT forms, one was ubiquitous, one was found in testis and liver, and a third was found in liver, kidney and thymus. No other phosphorylated sites were found, suggesting that the pI differences distinguishing between the various COMT forms are due to some as yet unidentified structural modification(s).

Utilising matrix-assisted laser desorption/ionisation techniques for the generation of structural information from different end-group functionalised poly(methyl methacrylate)s

End-group characterisation of three functionalised poly(methyl methacrylate) (PMMA) samples was performed by means of matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS). MALDI-time of flight (MALDI-TOF) data were employed to generate information about the initiators and chain-transfer agents used for the polymerisations. Further confirmation of the structural assignments was gleaned from MALDI-collision-induced dissociation (MALDI-CID) experiments. This latter technique is a powerful method for generating information on the masses of individual end groups.

TRPC3-interacting triadic proteins in skeletal muscle

The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation-contraction) coupling. However, it has not been possible to demonstrate direct protein-protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1-RyR1 and JP2-TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.

Identification of Mammalian Cell Lines Using MALDI-TOF and LC-ESI-MS/MS Mass Spectrometry

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.