matrix-assisted-laser-desorptionionization-time-flight-maldi-tof-mass

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 ...

Université J. Fourier

A method is presented for direct spatial analysis of amyloid beta peptides in biological tissue sections. The technique takes advantage of the very high sensitivity of matrix-assisted laser desorption/ionization mass spectrometry and is implemented on a commercial instrument with modifications to only a few components and the software. With this setup, hundreds of molecular images can be generated simultaneously and within just a few minutes. The current features are an instrumental resolution of 50 microm and a sensitivity down to the attomol range. This new technology is applied to the study of amyloid beta peptide distribution in brain sections from mice, showing features reminiscent of Alzheimer's disease.

Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry.

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F(430), a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F(430) in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F(430), variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F(430)M) in the (+)1, (+)2 and (+)3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle pi orbitals that are unique to Ni(I) forms of F(430). Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d-d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F(430) macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.

Spectroscopic investigation of the nickel-containing porphinoid cofactor F(430). Comparison of the free cofactor in the (+)1, (+)2 and (+)3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms.

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from naÃ¯ve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases.

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge.

2-D nanoscale LC combined with a triple quadrupole-linear ion trap mass spectrometer was applied to the analysis of a complex peptide mixture. A 2-D dual nanoscale LC-MS/MS system was compared to a conventional one. Peptides were separated with a strong cation exchange (SCX) microcolumn in the first dimension and two C18 nanocolumns were used as second dimension. MS experiments were performed using information-dependent data acquisition, where two precursor ions were selected from an enhanced MS (EMS) or an enhanced multicharged ion (EMC) as survey scan. The major benefit of EMC instead of EMS was a two-fold reduction of the data file and a 15% increase of characterized proteins. The advantage of the 2-D dual nanoscale LC-MS/MS system versus the conventional 2-D nanoscale LC-MS/MS system was reflected in the significant increase of peptides which were successfully identified within the same time frame. The first factor contributing to this increase was that the mass spectrometer was collecting twice the number of relevant MS/MS data. The second factor is the use of twice the number of SCX salt fractions in the first dimension, allowing a better sample fractionation, thereby reducing the number of peptides transferred to the second chromatographic dimension per salt fraction.

The application of 2-D dual nanoscale liquid chromatography and triple quadrupole-linear ion trap system for the identification of proteins.

A qualitative and quantitative analysis of erlotinib (RO0508231) and its metabolites was carried out on rat tissue sections from liver, spleen and muscle. Following oral administration at a dose of 5 mg/kg, samples were analyzed by matrix-assisted laser desorption ionization (MALDI) with mass spectrometry (MS) using an orthogonal quadrupole time-of-flight instrument. The parent compound was detected in all tissues analyzed. The metabolites following drug O-dealkylation could also be detected in liver sections. Sinapinic acid (SA) matrix combined with the dried-droplet method resulted in better conditions for our analysis on tissues. Drug quantitation was investigated by the standard addition method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the tissue extracts. The presence of the parent compound and of its O-demethylated metabolites was confirmed in all tissue types and their absolute amounts calculated. In liver the intact drug was found to be 3.76 ng/mg tissue, while in spleen and muscle 6- and 30-fold lower values, respectively, were estimated. These results were compared with drug quantitation obtained by whole-body autoradiography, which was found to be similar. The potential for direct quantitation on tissue sections in the presence of an internal standard was also investigated using MALDI-MS. The use of alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix resulted in better linearity for the calibration curves obtained with reference solutions of the drug when compared to SA, but on tissue samples no reliable quantitative analysis was possible owing to the large variability in the signal response. MS imaging experiments using MALDI in MS/MS mode allowed visualizing the distribution of the parent compound in liver and spleen tissues. By calculating the ratio between the total ion intensities of MS images for liver and spleen sections, a value of 6 : 1 was found, which is in good agreement with the quantitative data obtained by LC-MS/MS analysis.

Analysis of erlotinib and its metabolites in rat tissue sections by MALDI quadrupole time-of-flight mass spectrometry.

ALIX recruits ESCRT-III CHMP4 and is involved in membrane remodeling during endosomal receptor sorting, budding of some enveloped viruses, and cytokinesis. We show that ALIX dimerizes via the middle domain (ALIX(-V)) in solution. Structural modeling based on small angle X-ray scattering (SAXS) data reveals an elongated crescent-shaped conformation for dimeric ALIX lacking the proline-rich domain (ALIX(BRO1-V)). Mutations at the dimerization interface prevent dimerization and induce an open elongated monomeric conformation of ALIX(-V) as determined by SAXS modeling. ALIX dimerizes in vivo and dimeric ALIX colocalizes with CHMP4B upon coexpression. We show further that ALIX dimerization affects HIV-1 budding. C-terminally truncated activated CHMP4B retaining the ALIX binding site forms linear, circular, and helical filaments in vitro, which can be bridged by ALIX. Our data suggest that dimeric ALIX represents the active form that interacts with ESCRT-III CHMP4 polymers and functions as a scaffolding protein during membrane remodeling processes.

A crescent-shaped ALIX dimer targets ESCRT-III CHMP4 filaments.

Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0 degrees C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.

Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: plasmepsin 2 but not plasmepsin 4 is active under quenching conditions.

Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC(2)D complex, only in its reduced form (FADH(2)), which has the ability to reduce ferric iron. These results suggest that the SufBC(2)D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH(2)-dependent reductive mobilization of iron.

Iron-sulfur (Fe-S) cluster assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactor.

The H(+),K(+)-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an Î±-catalytic and a Î²-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H(+),K(+)-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the Î²-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,)(w) = 7.2 Â± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H(+),K(+)-ATPase Î±,Î²-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an Î±,Î²-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An Î±(2),Î²(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, Î±,Î² H(+),K(+)-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca(2+)-ATPase and Na(+),K(+)-ATPase.

Active detergent-solubilized H+,K+-ATPase is a monomer.

Let the Fur fly: Mutation of a single glutamate into an aspartate was shown to make the Fe sensor Fur as reactive to H2 O2 as the peroxide sensor PerR. In vivo and in vitro peroxide sensitivities of a series of PerR and Fur Asp/Glu mutants were studied by mass spectrometry. A combination of MÃ¶ssbauer spectroscopy analyses and DFT calculations gave a structural rationale for this behavior.

Luca Signor

Université J. Fourier

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