Xiaodan Tian

Implications of the serine protease HtrA1 in amyloid precursor protein processing

The defining features of the widely conserved HtrA (high temperature requirement) family of serine proteases are the combination of a catalytic protease domain with one or more C-terminal PDZ domains and reversible zymogen activation. Even though HtrAs have previously been implicated in protein quality control and various diseases, including cancer, arthritis, and neuromuscular disorder, the biology of the human family members is not well understood. Our data suggest that HtrA1 is directly involved in the β-amyloid pathway as it degrades various fragments of amyloid precursor protein while an HtrA1 inhibitor causes accumulation of Aβ in astrocyte cell culture supernatants. Furthermore, HtrA1 colocalizes with β-amyloid deposits in human brain samples. Potential implications in Alzheimers disease are discussed.

Isotope ratio and isotope dilution measurements using axial inductively coupled plasma time of flight mass spectrometry

Performance of axial ICP-TOF-MS in terms of precision and mass bias for isotope ratio measurements and accuracy of isotope dilution was assessed by measurements of isotopic reference materials for Li, Mg, Rb, Pt and Pb. Because of simultaneous ion extraction from the plasma, remaining flicker noise is minimized resulting in improved precision for isotope ratios compared to sequential mass spectrometers. Experimental relative standard deviations of <0.05% for isotope ratios were obtained at high signal levels measured in the analog detection mode. Effects on isotope ratios derived from changes of instrumental parameters, such as detector voltage and transverse rejection ion pulse settings, were evaluated. Isotope ratios for different concentrations changed when operating at too low detector voltages. An appropriate detector voltage setting is important but optimum voltages differ depending on the age and previous history of the detector. Results obtained for ID measurements of magnesium and rubidium amount content compared well with results from quadrupole ICP-MS and thermal ionization mass spectrometry. Mass bias per mass unit is high in the low mass range (13% forLi/Li) but around 0.2% in the high mass range. The long-term stability of ratios, measured discontinuously over several hours, is moderate; therefore measurements of an isotopic reference material at regular intervals is necessary to correct for small variations in mass bias over time.

QUALITY ASSURANCE OF ARSENIC, LEAD, TIN AND ZINC IN COPPER ALLOYS USING AXIAL INDUCTIVELY COUPLED PLASMA TIME-OF-FLIGHT MASS SPECTROMETRY (ICP-TOF-MS)

Results for As, Pb, Sn and Zn are reported for five copper alloys. Following dissolution in a refluxing HCl-HNO 3 mixture and subsequent dilution, the elements were determined by inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) using In as an internal standard. The following isotopes: 64 Zn, 66 Zn, 67 Zn, 68 Zn, 70 Zn, 75 As, 206 Pb, 207 Pb 208 Pb, 115 In, 116 Sn, 118 Sn and 120 Sn were monitored and the intensity ratios to 115 In as an internal standard were used. The analytical results were compared with results obtained by flame atomic absorption spectrometry (FAAS) for assessment of accuracy. The results agreed fairly well, except for Zn in some compositions, which was due to background interference effects, and for Sn, where dissolution problems occurred. The relative standard deviations, % RSDs, for six replicate measurements of the elements in each copper alloy were in the range 2.0-6.1% for As, 3.8-11.0% for Pb, 1.4-9.4% for Sn and 3.6-12.9% for Zn. Uncertainty budgets for Sn and Zn associated with these determinations are described. The detection limits (3s criterion) in the solid materials with a 1.0 g sample intake and appropriate dilutions were 0.7 µg g –1 for Pb, 2.5 µg g –1 for Sn, 11 µg g –1 for As and 15 µg g –1 for Zn.

Analytical performance of the microwave plasma torch in the determination of rare-earth elements with optical emission spectrometry

Abstract A recently developed microwave discharge device called the microwave plasma torch (MPT) is used in this work to evaluate its analytical performance in the determination of rare earth elements with optical emission spectrometry. Optimization of the instrument was performed by using individual elements with emphasis on the plasma observation zone, carrier and support gas flow rates. The detection limits for 15 rare earth elements obtained by MPT atomic emission spectrometry at 70 W are in the order of ng/ml. The precision (R.S.D.%) calculated with eleven continuous measurements at a concentration of about two orders of magnitude higher than the detection limits is in the range of 2.0 (Y) to 4.9 (Tb). Some comparisons were made between inductively coupled plasma and MPT methods, and the results show that in some cases, most of the sensitivity lines observed in these two sources for rare-earth elements are not identical. Compared with the ICP emission spectrum, the MPT source gives relatively simple spectra and much lower background emission. A preliminary examination of matrix effects with varying matrix concentrations shows that some easily ionized elements give significant enhancement on analyte signal and that aluminum suppresses the signals considerably. For some other elements (Co, Ni and Zn) no remarkable matrix effects are observed at a concentration level of 10 μg/ml.

Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry

Alzheimers disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in the brain that are directly involved in AD pathogenesis. The major component of AD plaques is ß-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, ß-and γ-secretase acting at the N- and C-terminal cleavage site, respectively. In this study, we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the γ-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Aß42 has been recently shown to be effective to inhibit and disaggregate Aß-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects as yet has been unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Aß(4–10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here, we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740–747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.

Helium direct current discharge ionization detector for gas chromatography

Abstract A novel helium direct current discharge ionization detector for gas chromatography (GC) has been developed. Two platinum electrodes are applied to sustain the discharge. Helium was used as both the GC carrier and the discharge support gas. The effects of configuration and operational conditions on the response characteristics of the detector were studied. The analytical performance of the detector for the determination of some permanent gases such as H 2 , N 2 , O 2 , Ar, CO, CO 2 and CH 4 was proved to be ideal. The detector has the advantages of high sensitivity, good stability, little dependence on the purity of helium, and extremely simple configuration and structure. The analytical figures of merit were shown to be comparable with those of the conventional helium ionization detector and the discharge ionization detectors. Also, the ionization mechanism of the detector was investigated. Furthermore, some practical sample analyses were done and the results were satisfactory.

Differential Epitope Identification of Antibodies Against Intracellular Domains of Alzheimer's Amyloid Precursor Protein Using High Resolution Affinity-mass Spectrometry

Several polypeptides comprising the carboxy-terminal domain of the 1-amyloid precursor protein (cAPP) were prepared by solid phase peptide synthesis, and employed as antigens for the determination of the epitopes recognised by anti-cAPP antibodies. Selective proteolytic epitope-excision and -extraction on the immobilised immune complexes, in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were used as major methods for epitope identification. The epitope recognised by a polyclonal anti-cAPP antibody (36-BO) was identified as APP(727-737), a sequence close to the APP transmembrane region. In contrast, the epitope recognised by a monoclonal anti-cAPP antibody (Jonas-mAb) was identified at APP(740-747) to be located more remote from the transmembrane region. The two adjacent, yet distinct epitopes recognised by two different antibodies should provide efficient tools for (i), molecular diagnostic applications, and (ii), the study of intracellular processing pathways of APP relevant to Alzheimers disease, utilising suitable mass spectrometric and molecular imaging approaches.

Synthesis, Structural Characterization and Reactivity Study of Humanin, an Alzheimer’s Disease Associated Peptide

Introduction Humanin (HN) is a novel 24 residue secretory peptide, detected in a normal region of human Alzheimer’s disease (AD) brain and shown to abolish in vitro neurotoxicity by all types of familial Alzheimer’s disease (FAD) mutants and by different -amyloid (A ) species. Therefore, HN represents a neuroprotective factor with therapeutic potential in AD [1]. However, mechanisms behind the rescue effects of HN in neuronal cells are still unclear. In this work, full-length HN was synthesized by solid phase peptide synthesis (SPPS) and the peptide homogeneity and amino acid sequence were confirmed by mass spectrometric measurements. HN secondary structure was characterized by circular dichroism spectroscopy (CD), NMR, and H/D exchange MS studies. Interaction of HN with synthetic A (1-40) peptide, highly involved in late-onset AD, was investigated using affinity chromatography in combination with high accuracy and resolution FTICR mass spectrometry. Preliminary results indicate a strong binding between HN and A (1-40) and allow a first delimitation of the recognition sequences, providing a new insight into understanding the in vivo function of HN.

Inductively Coupled Plasma Time of Flight Mass Spectrometry for Trace Element and Speciation Analysis

The main advantage of ICP-TOF-MS to previous ICP-MS instrumentation is the high speed and full elemental coverage in μl-volumes or rapid transients. For trace element determinations in samples from various biological systems the levels of several essential trace elements may interact and depend on each other in an intricate way. ICP- TOF-MS should be the method of choice if such inter-relationships are to be characterised since it incorporates unsurpassed speed and isotopic information allowing the use of stable isotope tracers. Moreover, coupling to chromatographic or laser ablation systems may lead to a better understanding of the trace element content in specific bands from a gel or a chromatographic peak adding even more selectivity to the analysis. The need for the TRIP is one of the weakest points of the present system and this feature is likely to be refined further in the future.