Erich R. Schmid

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface‐associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the eggs zona pellucida through a carbohydrate‐recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein‐chemical and mass spectrometric methods. AWN exists as two isoforms, AWN‐1 and AWN‐2, which differ in that AWN‐2 is N‐terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest‐neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN‐1 and AQN‐3, two other boar spermadhesins.

Fast screening method for the profile analysis of polycyclic aromatic hydrocarbon metabolites in urine using derivatisation–solid-phase microextraction

A method for the qualitative analysis of various metabolites of naphthalene, phenanthrene and pyrene is presented. The method uses SPME with a 85-microm polyacrylate fibre for extraction, headspace silylation with BSTFA without any catalyst for on-fibre derivatisation and GC-MS in the SIR mode for separation and detection. The suitability of the method for profile analysis of PAH metabolites is shown by analysing a smokers urine after enzymatic cleavage (and additionally after spiking with the target analytes) and spiked water. The method exhibits satisfactory separation of all investigated metabolites and no interferences due to matrix peaks.

High-performance liquid chromatographic-tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC-MS-MS. Linearity was given in the tested range of 50-5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50-5000 ng/ml was 1.47-6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37 degrees C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.

the amino acid sequence of AQN-3, a carbohydrate-binding protein isolated from boar sperm : location of disulphide bridges

Gamete recognition and adhesion are essential steps in fertilization. Among others, carbohydrate‐binding proteins on the sperm surface have been recognized to play a central role in the initial interaction of the male gamete with components of the zona pellucida of the homologous investing oocyte. We have isolated several members of a carbohydrate‐ and zona pellucida‐binding protein family from ejaculated sperm. Here we report the biological origin and structural characterization of AQN‐3, a component of this carbohydrate‐binding family. The molecular weight of purified AQN‐3 was determined by plasma desorption mass spectrometry. The protein was chemically and enzymatically degraded, the proteolytic fragments isolated and characterized by N‐terminal sequencing and fast atom bombardment mass spectrometry. In this manner we established the complete amino acid sequence of AQN‐3 and the location of its two disulphide bonds. No analogous protein sequence could be found in the MIPS protein sequence data bank, indicating that AQN‐3 may belong to a novel mammalian carbohydrate‐binding protein family.

QCM array for on-line-monitoring of composting procedures

Six QCM resonators forming a sensor array were coated with different molecularly imprinted polymers for the on-line monitoring of composting procedures. Four key analytes are traced, namely water, 1-propanol, ethyl acetate and limonene. Trendlines obtained on-line by the sensor during measurements in a commercial composter give a distinct pattern: the signal for the alcohols first decreases according to an increase in ethyl acetate concentration, and increases again, when obviously no more acetic acid is formed. Limonene is detected in later stages of composting. Similar trends could also be observed by GC-MS. Additionally, chromatographic and sensor data for limonene could be correlated with each other.

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm‐associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm‐associated and the seminal plasma inhibitors may confer to them different physico‐chemical properties which are postulated to be of functional importance.

Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues.

The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50 years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations ≥ 10.0 ng/μl were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72 h instead of 1-2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280 nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR).

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.

MALDI mass spectrometry of biomolecules and synthetic polymers using alkali hexacyanoferrate (II) complexes and glycerol as matrix

K4[Fe(CN)6]/glycerol and Na4[Fe(CN)6]/glycerol have been investigated as liquid matrix systems for UV-MALDI MS applying a N2 laser. Analyte molecules were detected as sodium or potassium adduct ions and, in the case of proteins, as well as protonated molecular ions. Mass accuracies were comparable to those found with standard solid matrix systems with −0.06 to +0.05% deviation in the reflectron mode and with −0.24 to +0.13% in the linear mode. Useful results could be obtained within a mass range of 15 000 Da for single-charged proteins and 8000 Da for potassium cationized polyethylene glycols. Detection limits were found for hydrophilic compounds in the low picomol range and for lipophilic compounds as triacylglycerols or peracetylated and partially benzylated carbohydrates in the low femtomol range. As shown by scanning electron microscopic investigations, the generation of a thin homogenous matrix layer was essential for a successful mass spectrometric experiment. A very careful cleaning of the target surface with glacial acid prior to matrix deposition improved the formation of such a matrix film that maximum sensitivity as well as good reproducibility of the experiments could be achieved.

Synthesis of pentasaccharide core structures corresponding to the genus-specific lipopolysaccharide epitope of Chlamydia

The trisaccharides allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->6)-O-2-aceta mid o-2-deoxy- beta-D-glucopyranosyl-(1-->6)-2-acetamido-2-deoxy-alpha- and -beta-D-glucopyranoside (16a and 16b), the tetrasaccharides allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->4)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->6)-O-2-aceta mid o-2-deoxy- beta-D-glucopyranosyl-(1-->6)-2-acetamido-2-deoxy-alpha- and -beta-D-glucopyranoside (19a and 19b), and the pentasaccharides allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->4)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->6)-O-2-aceta mid o-2-deoxy-bet a -D-glucopyranosyl-(1-->6)-2-acetamido-2-deoxy-alpha- and -beta-D-glucopyranoside (23a and 23b) were prepared. The glycosidic linkages were formed using 1,3,4,6-tetra-O-acetyl-2-chloroacetamido-2-deoxy-beta-D-glucopy ran ose (6) and FeCl3 as promoter as well as per-O-acetylated Kdo mono- and di-saccharide bromide derivatives (12 and 20) under Helferich conditions. The oligosaccharides, which correspond to dephosphorylated part-structures of enterobacterial and chlamydial lipopolysaccharides, were characterized by NMR spectroscopy as well as plasma desorption and matrix-assisted laser desorption mass spectrometry.