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For metabolite profiling chemical derivatization has been used to improve MS sensitivity and LC retention. However, for multi-analytes quantification, the number of commercially available isotopically labelled internal standards is limited. Besides, there is no single workflow which can provide large-scale metabolomics coverage in particular for polar metabolites. To overcome these limitations and to improve reproducibility a fully automated dual derivatization approach was developed. Differential Isotope Labeling (DIL) was adopted by derivatizing carbonyl, amino and phenol metabolites with two isotopic forms. Urine samples were derivatized with 12C-dansyl chloride (DnsCl) and 12C-dansylhydrazine (DnsHz). Suitable quantification standards were generated by derivatized 40 standards including amino acids, sex hormones and other highly polar metabolites with labelled 13C2-dansyl chloride and 13C2-dansylhydrazine. The derivatization of the standards and the urine sample was performed using a PAL RTC autosampler in-line to column-switching LC-HRMS analysis with data independent acquisition (SWATH-MS). The parallel reactions were completed in 15 minutes inside of two agitators at different conditions overlapping with the LC-MS analysis time which was of 25 minutes. The column switching setup is critical to remove the excess of reagents which can negatively affect the ionization efficiency and deteriorate the chromatographic performance. The combination of dual DIL with SWATH-MS acquisition enables post-identification of unknown metabolites and quantitation at precursor (MS1) and specific tag fragment (MS2) levels. The inter- and intra-batch accuracy and precision of the method fall in the range ± 15% using single point calibration, and at MS1 or MS2 level providing full flexibility. The method was successfully applied to the analysis of human urine samples.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

バリヤー放電ラジオ波ヘリウムプラズマ発光法によるフッ素, 塩素, 臭素及びヨウ素を定量する方法を開発した. ヘリウムガスを通じたガラス管の外側に2枚の銅はくを巻き付け, 片方を接地し, 他方にラジオ波インパルス高電圧 (98kHz, 3.2kV) を与え, 放電を起こした. 放電から発せられる光を光ファイバーを介して, 電荷結合素子 (CCD) 付き分光器に導入し, 730～960nmの領域の光を測光した. フッ素 (733.2, 739.9nm), 塩素 (833.3, 837.6, 858.6, 894.8, 912.1nm等), 臭素 (827.2, 882.5, 889.8, 926.5nm等) 及びヨウ素 (905.8nm) の原子線が観測され, フッ素, 塩素については1～10μg, 臭素については0.1～1μgの範囲で, 量-応答の直線性が確認された. 各元素の発光強度を比較したところ, 臭素>ヨウ素>塩素>フッ素の順番であった.

In terms of resolution, mass accuracy, and sensitivity, the Orbitrap represents one of the most potent mass analyzers available today. We here elucidate the potential of interfacing Orbitrap-MS to ion-pair RP HPLC for intact protein analysis. Using gradients of ACN and monolithic columns of 1.0 and 0.10 mm id, peak capacities between 120 and 130 were achievable within 20-25 min separation time. Compared with silica-based stationary phases, protein recovery and carryover from monolithic columns were found clearly superior. Intact proteins were detectable in a mass range covering 5.7-150 kDa with LODs in the low femtomol range. Compared with UV detection, MS detection with a scanning speed of 1.6 s per spectrum on average led to a 26% increase in chromatographic peak widths, whereas chromatographic patterns were mostly preserved in extracted ion chromatograms at an acquisition rate of 0.5 s per spectrum. Isotopic resolution of multiply charged ions was demonstrated for proteins up to 42 kDa. A micro-HPLC-Orbitrap-MS setup employing a 1.0 mm id column was utilized to characterize a 150 kDa recombinant monoclonal antibody. The applicability of nano-HPLC-Orbitrap-MS to the analysis of highly complex protein mixtures is demonstrated for the 70% ethanol extractable subproteome of wheat grains.

Usually it is difficult to achieve good absorption of microwave power in plasmas of small dimension and low collision frequency if no external magnetic field is superimposed. If the plasma frequency is less than the frequency of the electromagnetic wave, e.g. the plasma density is less than the so-called critical density, the plasma is rather transparent. If the density becomes higher than the critical density, the plasma behaves more and more as a good reflector. In both cases only a small fraction of the incident power is absorbed. The poor absorption in the overdense plasma is caused by a pronounced skin effect.

Measured high and low temperature bremsstrahlung isochromats of Pd and Rh are reported and compared with free electron densities of states derived from γ- and χ-measurements. The Pd- as well as the Rh-isochromats do not show any significant dependence on the target temperature except the temperature dependent smearing out of the Fermi edge at high temperatures. From this result we conclude, that isochromats—in contrast to γ- and χ-measurements—are not influenced by enhancement effects.