Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.     

Sample preparation of human serum for the analysis of tumor markers. Comparison of different approaches for albumin and gamma-globulin depletion

LC-MS is a powerful method for the sensitive detection of proteins and peptides in biological fluids. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomic studies. In human serum the most abundant proteins are albumin and gamma-globulins. We tested several approaches to specifically reduce the level of these proteins based on either specific antibodies, dye ligands (for albumin) and protein A or G (for gamma-globulins). The resulting, depleted serum was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and LC-MS for the residual presence of these abundant proteins as well as for other serum proteins that should remain after depletion. To test the applicability of this method to real-life samples, depleted serum of a cervical cancer patient was analyzed for the presence of a specific tumor marker protein SCCA1 (squamous cell carcinoma antigen 1; P29508), which is present at ng/ml concentrations. The results demonstrate that SCCA1 can be detected by LC-MS in patient serum following depletion of albumin and gamma-globulins thus opening the possibility of screening patient sera for other, so far unknown, tumor markers.     

Chiral hydroperoxides as oxygen source in the catalytic stereoselective epoxidation of allylic alcohols by sandwich-type polyoxometalates: control of enantioselectivity through a metal-coordinated template

The epoxidation of allylic alcohols is shown to be efficiently and selectively catalyzed by the oxidatively resistant sandwich-type polyoxometalates, POMs, namely [WZnM(2)(ZnW(9)O(34))(2)](q)(-) [M = OV(IV), Mn(II), Ru(III), Fe(III), Pd(II), Pt(II), Zn(II); q = 10-12], with organic hydroperoxides as oxygen source. Conspicuous is the fact that the nature of the transition metal M in the central ring of polyoxometalate affects significantly the reactivity, chemoselectivity, regioselectivity, and stereoselectivity of the allylic alcohol epoxidation. For the first time, it is demonstrated that the oxovanadium(IV)-substituted POM, namely [ZnW(VO)(2)(ZnW(9)O(34))(2)](12-), is a highly chemoselective, regioselective, and also stereoselective catalyst for the clean epoxidation of allylic alcohols. A high enantioselectivity (er values up to 95:5) has been achieved with [ZnW(VO)(2)(ZnW(9)O(34))(2)](12)(-) and the sterically demanding TADOOL-derived hydroperoxide TADOOH as regenerative chiral oxygen source. Thus, a POM-catalyzed asymmetric epoxidation of excellent catalytic efficiency (up to 42 000 TON) has been made available for the development of sustainable oxidation processes. The high reactivity and selectivity of this unprecedented oxygen-transfer process are mechanistically rationalized in terms of a peroxy-type vanadium(V) template.     

Calculation of cross sections for positron-Ar collisions

The three-body classical trajectory Monte-Carlo (CTMC) method is used to investigate positron-argon atom collisions. The total ionization cross sections are presented along with singly and doubly differential cross sections. The existence of the cusp-like peak in the triply differential electron and positron spectra at positron impact is predicted.     

Cluster aggregation: a new method for producing atomic and molecular clusters

A new method for the production of cold free clusters is presented. A beam of large rare gas clusters is passed through a low pressure atomic gas. The gas atoms are picked up by the rare gas clusters whereby they condense. A complete evaporation of rare gas atoms from the newly formed clusters then follows once the number of captured particles becomes sufficiently large. The method is demonstrated for Xe clusters with size up to 500 atoms per cluster but it should work for most materials, including the refractory materials.     

Franck-Condon matrix elements for bound-continuum vibrational transitions calculated by numerical integration and basis set expansion techniques

Franck-Condon factor distributions for bound-to-continuum transitions of one-dimensional vibrational states are calculated by a) using numerical integration, b) employing a finite number of square integrable harmonic oscillator functions. The methods are generally applicable to any kind of bound or repulsive potential involved. Results are presented and compared to model potential calculations previously reported by Krüger [1].     

Intramolecular multiple scattering in electron diffraction from fluoro-methyl halides

Relative elastic differential cross sections for scattering of 1 keV electrons from CF₃H, CF₄, CF₃Cl, CH₃F and CH₃Cl molecules were measured for momentum transfer from 3 Å^?1 to 29 Å^?1. The oscillatory residuals remaining after substraction of the atomic scattering and the molecular interference contributions calculated in the independent atom model are ascribed to the influence of intramolecular multiple scattering. The data reflect the dominant contributions of the CF₃-group and show larger oscillations than obtained from triple scattering calculations according to the method of Liu and Bonham using the second Born approximation.     

Preparation of optically active allylic hydroperoxy alcohols and 1, 3-diols by enzyme-catalyzed kinetic resolution and photooxygenation of chiral homoallylic alcohols

All four possible enantiomers of the 3-hydroperoxy-4-penten-1-ols 2a, b and their corresponding 4-pentene-1,3-diols 4a,b have been prepared for the first time in high enantiomeric purity (up to 98% ee) and in preparative amounts according to two distinct ways: First the photooxygenation of the racemic homoallylic alcohols 1 gave the racemic hydroperoxy alcohols 2, which have subsequently been kinetically resolved by horseradish peroxidase (HRP); alternatively, first the lipase-catalyzed resolution afforded the optically active homoallylic alcohols 1 and subsequent photooxygenation led to the enantiomerically enriched hydroperoxy alcohols 2.     

Oxidation of micro or trace amounts of bromide to bromate by peroxodisulphate before their iodometric determination by titration or spectrophotometry

The optimum conditions for the oxidation of bromide to bromate by peroxodisulphate at 120 degrees as well as for the decomposition of the excess of oxidant have been determined. The predicted advantages of this oxidizing agent, viz. minimal blanks and destruction of small amounts of interfering organic matter and reducing substances, were confirmed. The bromate was determined iodometrically either by titration with thiosulphate or by spectrophotometry in absence of oxygen at 355 nm. The titrimetric finish applied to 0.8-8 micromole of bromide gave a mean yield of 100.0%, s = 6 nmole. The spectrophotometric finish applied to 0.05-0.25 micromole of bromide gave a mean yield of 98.9%, s = 1.1 nmole. Interfering amounts of iodide present in the sample and oxidized to iodate can be corrected for by making use of the pH-dependence of the reaction of iodide with bromate and iodate.