Position of the Peptide Linkage in Glutamyl-Taurine from Calf Brain Synaptic Vesicles

To elucidate the position of the peptide bond in glutamyl-taurine this dipeptide was extracted from calf brain synaptic vesicles and subjected to paper electrophoresis. It was analyzed further in an automatic amino acid analyzer prior and subsequent to acid hydrolysis. Both alpha- and gamma-forms were found to be present in approximately equal amounts.     

Mass Spectrometric Analyses of Brain Synaptic Peptides Containing Taurine

The structure of a number of low-molecular-weight acidic peptides containing taurine prepared from calf brain synaptosomes and their subcellular vesicles was studied using electron impact mass spectrometry. At least seven sequences could be identified: N-acetylaspartyl-glutamyl-taurine, N-acetylaspartyl-taurine, N-acetylglutamyl-taurine, glutamyl-taurine, aspartyl-taurine, seryl-glutamyl-seryl-taurine, and seryl-taurine.     

Using cyclic peptide mixtures as probes for metal ion host-guest interactions

Summary Mixtures of cyclic peptides, formed by head-to-tail cyclizations of side-chain resin-bound linear sequences, have been prepared using solid-phase synthesis. Fast atom bombardment mass spectrometry of cyclic peptides with various metal ions can reveal preferred modes of host-guest patterns, albeit in a nonquantitative manner. This approach could prove useful for more rapid screening of potential peptide ionophores. A cyclic heptapeptide with a dipeptide tail proved to be a particularly effective host for a Ca²⁺ ion; in a small three-component mixture, cyclo[Gly-Asp-d-Pro-Xxx-Asp-d-Pro-Asp(Aca-Phe-NH₂)], binding to Ca²⁺ varied from Xxx=N-MeAla>GlySar. In a 15-component mixture, cyclo[Pro-Xxx-Asn-Pro-Xxx-Asn] where Xxx=Ala, Glu, Leu, Lys or Phe, there were no significant differences with respect to binding to metal ions. We believe this to be the first reported use of cyclic peptide libraries for screening metal ions to discern host-guest relationships.Abbreviations Aca aminocaproic acid - Boc tert-butyloxycarbonyl - BOP benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate - DCM dichloromethane - DIEA diisopropylethylamine - DMF N,N-dimethylformamide - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - pMBHA 4-methylbenzhydrylamine - TFA trifluoroacetic acid This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Fast atom bombardment: A new mass spectrometric method for peptide sequence analysis

We have studied a selection of peptides using a new mass spectrometric ionisation technique - fast atom bombardment (FAB). We define the fragmentation pathways observed and comment on the utility in sequence analysis. A simple acetylation experiment is shown to aid rapid sequence assignment.     

快原子轰击质谱技术在异常血红蛋白一级结构分析中的应用

本文报道用快原子轰击(FAB)质谱技术测定血红蛋白(Hb)肽链经胰蛋白酶水解后的几个片段的氨基酸顺序结果。通过解释正离子谱上的碎片峰,确证用FAB质谱测得的氨基酸顺序与已知的完全一致。在此基础上,我们对二例未知结构的异常Hb进行测定,确证一例是Hb G Taipei(α_2β_2~(22Glu→Gly)),另一例是HbG Honolulu(α_2~(30Glu→Gln)β_2)。实验表明,用FAB质谱技术测定蛋白质一级结构具有微量、快速、可靠和操作简便等优点。     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

Brain Gangliosides of Quaking and Shiverer Mutants: Qualitative and Quantitative Changes of Monosialogangliosides in the Quaking Brain

Ganglioside compositions in the brains of the mutant mice quaking and shiverer were compared with those of their littermate controls, C57BL/6 and C3HSWV. Neither ganglioside content nor composition of shiverer brains differed from those of the control brains. Change in the ganglioside composition of the mutant brain from that of the control was observed only in the quaking mutant brain, in which monosialoganglioside GM1 was significantly reduced and GM4 was completely absent. The structures of the gangliosides were determined by negative ion fast atom bombardment mass spectrometry, and the GM3 and GM4 gangliosides in the quaking brain were found to be altered in regard to their long-chain base and fatty acid compositions when compared to the normal C57BL/6 brain.     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

Fatty acid profiling of phospholipids by field desorption and fast atom bombardment mass spectrometry

Natural phosphatidylcholines, phosphatidylethanolamines and sphingomyelins have been investigated by field desorption and fast atom bombardment mass spectrometry. It is demonstrated that using these soft mass spectrometric ionization techniques, accurate, fast, and sensitive fatty acid profiling of phospholipids can be performed. With respect to the analysis of intact molecular species both ionization techniques reveal similar results. Using field desorption, a specific fragment ion provides a fast access to the total distribution of fatty acids in complex lipids. Generally, a good agreement between the mass spectrometric abundance data and those produced by gas chromatographic analysis is observed.     

Fast atom bombardment mass spectrometry of the anthocyanins violanin and platyconin

Fast atom bombardment mass spectrometry (FABMS) of the acylated anthocyanin violanin confirmed its molecular mass (919) and gave a fragmentation pattern in agreement with its known structure. FABMS of the more complex flower pigment platyconin gave its accurate molecular mass as 1421.3740 and a fragmentation pattern suggesting that platyconin is delphinidin 3-rutinoside-5-glucoside with two mols of glucosylcaffeic acid attached to the glucose moiety of rutinose in a branched-chain arrangement.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.     

快原子轰击质谱技术结合酶学方法鉴定黑曲霉1,6—缩水—β—D—吡喃葡萄糖激酶的诱导合成

1,6－缩水－β－D－吡喃葡萄糖是纤维素类物质热解的主要产物,黑曲霉突变株CBX－209能较好地利用该糖作为唯一的碳源和能源生长并产生有用的代谢产物柠檬酸,其效率与利用葡萄糖大致相当。利用葡萄糖氧化酶和竦根过氧化物酶复合系统测定证明该菌株不存在1,6－缩水－β－D－吡喃葡萄糖水解酶,采用快原子轰击质谱技术结合6－磷酸葡萄糖脱氢酶系统进行测定,结果表明经（NH4）2SO4沉淀或阴离子交换层析析处理后的无细胞提取液在加入ATP和Mg^2 条件下能直接催化1,6－缩水－β－D－吡喃葡萄糖合成6－磷酸葡萄糖,证明黑曲霉突变株中存在一个新酶,即1,6－缩水－β－D－吡喃葡萄糖激酶。该酶为诱导酶。     

Isoprenoid quinones in an aerobic hyperthermophilic archaeon, Aeropyrum pernix

Aeropyrum pernix is the first strictly aerobic hyperthermophile known to grow heterotrophically at neutral pH and at temperatures up to 100°C. Using a simple and sensitive frit-fast atom bombardment liquid chromatography/mass spectrometry quinone analysis method, we analyzed the quinones in A. pernix. This organism contained demethylmenaquinone analogs (DMK-6(H_n)) and methionaquinone analogs (MTK-6(H_n)) when it was grown under vigorous shaking in the presence of air. The quinones were partially or fully saturated with six isoprenyl units. Although DMK and MTK are the quinones found in eubacteria, this is the first report to demonstrate the simultaneous occurrence of DMK and MTK in archaea. The effect of Na₂S₂O₃ on the quinone composition was studied at concentrations of 0, 0.1 and 0.5% under aerobic growth conditions with shaking. The total quinone content was highest (83.4 μg g⁻¹ dry cell weight) at 0.1% Na₂S₂O₃. In the absence of Na₂S₂O₃, only DMK-6 analogs were detected. While DMK analogs such as DMK-6(H₁₂), DMK-6(H₁₀) and DMK-6(H₈) were the major quinones at 0.1% Na₂S₂O₃, MTK analogs such as MTK-6(H₁₂) and MTK-6(H₁₀) were also detected. When the Na₂S₂O₃ concentration was increased to 0.5%, both DMK-6(H₈) and MTK-6(H₁₀) disappeared, while MTK-6(H₁₂) increased to approximately 20% of the total quinone content. When A. pernix was grown under oxygen limitation in a tightly closed bottle without gas phase, MK-6(H₁₀) appeared.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.     

1-trans-Parinaroyl phospholipids: Synthesis and fast atom bombardment/electron impact mass spectrometric characterization

1-trans-Parinaroyl-2-linoleoyl-sn-glycero-3-phosphocholine (1–18:4-2-18:2-GPC) was synthesized from lecithin and parinaric acid by the following route: $\text{diacyl-GPC}\text{→}\text{GPC}\text{→ 1,2-}\text{di}\text{-18:4-}\text{GPC}\text{(}\text{I}\text{) → 1–18:4-}\text{GPC}\text{(}\text{II}\text{) → 1–18:4-2-18:2-}\text{GPC}\text{(}\text{III}\text{)}$. The identity of I, II and III was established by fast atom bombardment (FAB) mass spectrometry of the intact molecules as well as electron impact (E1) mass spectrometry of the corresponding O-TMS derivatives obtained after phospholipase C treatment and silylation. Temperature dependent phase transition of phospholipid liposomes was performed in the presence of III.     

Mass spectrometric studies of underivatized polyphenolic glycosides

Flavonoid and xanthone glycosides were studied by mass spectrometry using soft ionization techniques such as desorption chemical ionization, fast atom bombardment and field desorption. In all cases a preliminary derivatization was not required. Information on the M_r, the sugar sequence and structure of the aglycone could be obtained.