An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low‐energy collision‐induced dissociation tandem mass spectrometry (MS/MS), mainly long b‐fragments are observed, limiting the possibility to determine variations such as amino acid variants or post‐translational modifications (PTMs) within the N‐terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann–Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O‐glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top‐down MS‐based method. Copyright © 2012 John Wiley & Sons, Ltd.     

The Applicability of Enzymes in Cellulose Ether Analysis

Summary: Six methyl cellulose (MC) samples, one with a DS of 1.32 and five with a DS between 1.83 and 1.88, were degraded with five different enzymes or enzyme preparations containing endoglucanases. The main goal was to investigate whether enzymes could be used for determination of heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity it was necessary to gather information on how the enzymes affect hydrolysis. Monomer composition and methyl distribution in the polymer chain were analyzed after total or partial random hydrolysis and appropriate derivatization by GC and MS, respectively, and used as reference data for the evaluation of the enzymatic hydrolysis. Size exclusion chromatography with multi angle light scattering and refractive index detection (SEC-MALLS/RI) was used to estimate molar mass distribution of the MCs before and after hydrolysis. Electrospray and matrix assisted laser desorption/ionization (ESI and MALDI) in combination with various MS analyzers were compared with respect to quantification of the degradation products directly and after perdeuteromethylation. Methyl group distribution in the oligomeric fractions and the average DS/DP were calculated from ESI mass spectra. With help of the reference analysis, patterns could be corrected for the unspecific contribution of end groups. By labelling and ESI-MSⁿ, our knowledge about the tolerance of the enzyme's sub-sites with respect to the number of methyl groups could be improved. A novel standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-IT MS) was used to determine the amount of formed oligomers.     

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised methyl cellulose

Methyl cellulose (MC) was partially depolymerised and the oligomers thus obtained were studied by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised MC were collected from size-exclusion chromatography and subjected to a sample preparation investigation. Several MALDI matrices and solvents were evaluated. The results showed that the solvent choice had a significant effect on the measured degree of substitution (DS). Aprotic solvents produced higher DS values, which was most likely due to poor solubility of species with low DS. The obtained signal intensity, however, did not correlate with the solubility but seemed to be more dependent on certain matrix/solvent combinations. All the matrices attempted produced mass spectra with sufficient signal intensity for accurate peak area calculation. The choice of matrix did not have any significant effect on the measured DS. Sample spots obtained from organic solvents had a more homogeneous distribution of the analyte and smaller crystals than those obtained from water. This increased both the reproducibility and peak resolution and in addition the analysis time was shorter. DS measurements were performed on two acidically depolymerised MCs with different nominal DS values. It was easy to distinguish between the two MCs, and the measured DS values agreed well with the values supplied by the manufacturers.     

Comprehensive analysis of the substituent distribution in hydroxyethyl celluloses by quantitative MALDI-ToF-MS

Three HECs with a high MS (HEC 1: 1.89, HEC 2: 1.94, HEC 3: 3.03) were analyzed with respect to their substituent distribution and tandem reaction in the glucosyl unit by GLC of the corresponding glucitol acetates, and along the polymer chain by MALDI-ToF-MS after a multi-step sample preparation. For comparison of the experimental data with a random pattern an extended Bernoulli plot was applied to calculate a random distribution for the composition of un-, mono-, di-, tri-, and up to heptasubstituted glucosyl units (c0, c1, c2, ... c7).     

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) of partially depolymerised carboxymethyl cellulose (CMC) have been investigated. The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised CMC were collected from size-exclusion chromatography (SEC) and further investigated by MALDI-TOFMS. 2,5-Dihydroxybenzoic acid was used as matrix, dissolved in H(2)O due to the poor solubility of CMC in suitable organic solvents. The samples were dried by two methods, in ambient atmosphere and at reduced pressure. Under reduced pressure the sample spot homogeneity increased. This drying method, however, produced additional adduct peaks in the mass spectra originating from ion exchange on the CMC oligomers. Analysis of CMC could be performed in both negative and positive ion modes. Mass discrimination and variation in ionisation efficiency were demonstrated by comparing mass spectra with SEC data. Measurements of the degree of substitution (DS) were performed on three CMCs with different DS values, which were depolymerised in trifluoroacetic acid. The three CMCs were easily distinguished from one another, but the obtained DS values deviated from the values supplied by the manufacturer.     

Improved matrix-assisted laser desorption/ionisation sample preparation of a partially depolymerised cellulose derivative by continuous spray deposition and interfacing with size-exclusion chromatography

Continuous spray deposition (CSD) of aqueous solutions of partially depolymerised methyl cellulose was found to improve matrix-assisted laser desorption/ionisation (MALDI) sample preparation. One feature was that the sensitivity in MALDI time-of-flight mass spectrometry increased up to an order of magnitude compared with the standard sample preparation method. Another feature was that CSD provided targets for MALDI with homogeneously distributed analyte. This resulted in a more even signal intensity and a higher reproducibility than in the standard method. High-mass discrimination was more pronounced in CSD than in the standard method. Size-exclusion chromatography with aqueous eluent was coupled online to CSD onto matrix-precoated foils. The suitability for determination of the molar mass distribution of methyl cellulose was investigated.     

An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.