Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) serves as a rapid and accurate means to determine masses of proteins independent of their shapes or interactions with other molecules. It provides one of the most fundamental characterizations of major plasma proteins. Purified proteins in saline or serum specimens were prepared for analysis by dilution, mixing with a solution of sinapinic acid, and drying on a target plate. Specimens were analyzed in a linear TOF mode with external calibration. Analyses of 24 purified plasma proteins showed predominance of singly charged ions with lesser amounts of dimer and doubly charged monomer, and provided measured masses for these proteins. A number of proteins, including albumin, transferrin, apolipoproteins A-I, A-II, C-I, C-II, and C-III, and prealbumin, could be analyzed directly in serum with appropriate dilution. Measured values for masses of major plasma proteins will assist in analysis of serum and plasma. It is possible to analyze a number of components by MALDI-TOF/MS directly in diluted serum. Extremely simple sample preparation techniques may be useful in analyzing structural variation of several major plasma proteins, particularly those with masses <30 kDa, including a number of apolipoproteins and markers of nutritional status or acute phase responses.     

Fermentation of pectin by a newly isolated Clostridium thermosaccharolyticum strain

Abstract By enrichment on pectin a thermophilic anaerobic bacterium was isolated. This strain, identified as Clostridium thermosaccharolyticum , was capable of fast growth on pectin (μ_max 0.58 h⁻¹) forming acetate, butyrate, hydrogen, carbon dioxide, methanol and traces of ethanol. The optimum temperature for growth was 58°C and the optimal pH was 6. The initial breakdown of pectin was catalysed by methylesterase and polygalacturonate hydrolase activity; no polygalacturonate lyase activity was found.     

Chemical characterization of the regularly arranged surface layers of Clostridium thermosaccharolyticum and Clostridium thermohydrosulfuricum.

Clostridum thermosaccharolyticum and Clostridium thermohydrosulfuricum possess as outermost cell wall layer a tetragonal or hexagonal ordered array of macromolecules. The subunits of the surface layer can be detached from isolated cell walls with urea (8M) or guanidine-HCl (4 to 5 M). Triton X-100, dithiothreitol, ethylenediaminetetracetate, and KCl (3 M) had no visible effect on the regular arrays. Sodium dodecyl sulfate-polyacrylamide electrophroesis showed that, in both organisms, the surface layer is composed of glycoprotein of molecular weight 140,000. The glycoprotein from both microorganisms has a predominantly acidic amino acid composition and an acidic isoelectric point after isoelectric focusing on polyacrylamide gels. The glycocomponent is composed of glucose, galactose, mannose, and rhamnose.     

Analysis of the Anaplasma marginale major surface protein 1 complex protein composition by tandem mass spectrometry

The protective major surface protein 1 (MSP1) complex of Anaplasma marginale is a heteromer of MSP1a and MSP1b, encoded by a multigene family. The msp1beta sequences were highly conserved throughout infection. However, liquid chromatography-tandem mass spectrometry analysis identified only a single MSP1b protein, MSP1b1, within the MSP1 complex.     

Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70

Clostridium thermosaccharolyticum D120-70 possesses as its outermost cell envelope layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures: (formula; see text).     

Lipid specificity of surfactant protein B studied by time-of-flight secondary ion mass spectrometry

One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.     

Accurate mass analysis of phosphoramidites by electrospray mass spectrometry

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.     

Reconstitution of rapeseed high molecular weight protein from isolated subunits

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide-gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10 mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.     

A lithotrophic Clostridium strain with extremely thermoresistant spores isolated from a pectin-limited continuous culture of Clostridium thermosaccharolyticum strain Haren

Abstract A thermophilic Clostridium sp. with extremely thermoresistant spores was isolated from a pectin-limited continuous culture of Clostridium thermosaccharolyticum . The decimal reduction time of the spores was 70 min at 121°C. Because of the ability of the bacterium to grow both heterotrophically and lithotrophically, it has a potential for infecting a wide range of thermophilic anaerobic cultures.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

Molecular weight determination of plasmid DNA using electrospray ionization mass spectrometry.

Ionization and molecular weight (MW) determination of megadalton size plasmid DNA has been achieved using electrospray ionization (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. DNA molecules were shown to remain intact through electrospray ionization by collection on a specially prepared surface, followed by agarose gel electrophoresis. Individual highly charged ions of plasmid DNA produced by ESI were trapped in an FTICR cell for up to several hours and reacted with acetic acid to induce charge state shifts. Measurements of mass-to-charge ratios for these multiple peaks arising from charge state shifting give MW measurements of individual ions with an average accuracy of 0.2%. The MW distribution was obtained by measurements for a number of individual ions from the same sample [plasmid DNA: pGEM-5S MW(cal) = 1.946 MDa], yielding a MW(obs) of 1.95 +/- 0.07 MDa for ions clustered in the vicinity of the expected MW.     

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry

A method for the quantification of acetylpolyamines, N¹,N¹²-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N¹,N⁸-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using ¹⁵N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of α,ω-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N¹-acetylspermidine and N⁸-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).     

Desmocalmin: a calmodulin-binding high molecular weight protein isolated from desmosomes

A unique high molecular weight protein (240,000 mol wt) has been purified from isolated desmosomes of bovine muzzle epidermis, using low-salt extraction at pH 9.5-10.5 and gel-filtration followed by calmodulin-affinity column chromatography. This protein was shown to bind to calmodulin in a Ca2+-dependent manner, so we called it desmocalmin here. Desmocalmin also bound to the reconstituted keratin filaments in vitro in the presence of Mg2+, but not to actin filaments. By use of the antibody raised against the purified desmocalmin, desmocalmin was shown by both immunoelectron and immunofluorescence microscopy to be localized at the desmosomal plaque just beneath the plasma membrane. Judging from its isoelectric point and antigenicity, desmocalmin was clearly distinct from desmoplakins I and II, which were identified in the desmosomal plaque by Mueller and Franke (1983, J. Mol. Biol., 163:647-671). In the low-angle, rotary-shadowing electron microscope, the desmocalmin molecules looked like flexible rods approximately 100-nm long consisting of two polypeptide chains lying side by side. The similar rodlike structures were clearly identified in the freeze-etch replica images of desmosomes. Taken together, these findings indicate that desmocalmin could function as a key protein responsible for the formation of desmosomes in a calmodulin-dependent manner (Trinkaus-Randall, V., and I.K. Gipson, 1984, J. Cell Biol., 98:1565-1571).     

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.     

Mutation-tolerant protein identification by mass spectrometry.

Database search in tandem mass spectrometry is a powerful tool for protein identification. High-throughput spectral acquisition raises the problem of dealing with genetic variation and peptide modifications within a population of related proteins. A method that cross-correlates and clusters related spectra in large collections of uncharacterized spectra (i.e., from normal and diseased individuals) would be very valuable in functional proteomics. This problem is far from being simple since very similar peptides may have very different spectra. We introduce a new notion of spectral similarity that allows one to identify related spectra even if the corresponding peptides have multiple modifications/mutations. Based on this notion, we developed a new algorithm for mutation-tolerant database search as well as a method for cross-correlating related uncharacterized spectra.     

Molecular weight determination of methyl esters of mycolic acids using thermospray mass spectrometry.

Methyl esters of normal fatty acids, corynomycolate and corynomycolenate were used as model compounds for thermospray mass spectrometric procedures for molecular weight determination of the related nocardial mycolic acids. By using ammonium acetate at the positive ion generator, in both cases, a family of ions was produced. The following members were found and corresponded to the adducts: (1) M + H; M + NH4 and M + H + NH4 for methyl esters of normal fatty acids, whereas M + H, M + 2H and M + H + NH4 were the adducts most frequently observed with methyl corynomycolates. The methyl esters of C40-C48 mycolic acids from Rhodococcus rhodochrous exhibited prominent peaks corresponding to adducts M + H + NH4 whereas those corresponding to M + 2H showed slightly lower intensities. The structure M + H had no significant representatives with this subclass of mycolic acids. A similar pattern was observed with methyl esters of C50-C54 mycolic acids from Nocardia asteroides GUH-2. Ion peaks C50-C54 representing adducts M + 2H and M + H + NH4 prevailed in the mass spectrum. In this case, the intensities of peaks corresponding to M + 2H were slightly higher than those of the M + H + NH4. Essentially three main species of nocardomycolic acids were detected: (1) monounsaturated C50:1, C52:1 and C54:1; (2) diunsaturated C50:2, C52:2 and C54:2 and (3) triunsaturated C52:3 and C54:3 mycolic acids. The most abundant mycolic acid was C52:2 followed in decreasing abundance by C52:1, C54:2, C50:2, C52:3 and C54:3 mycolic acids.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.