A novel method of peptide sequencing by mass spectrometry is described. Metastable decay of laser-desorbed ions, taking place in the first field-free drift region of a reflection time-of-flight mass spectrometer, has been monitored to get structural information from larger peptides. Fragment ions from metastable decay are mass analysed by adjusting the potentials of the ion reflectron according to the kinetic energies of the ions. The features of the technique and its significance for future applications are outlined.

Fragmentation of protein and peptide ions generated by matrix-assisted laser desorption has been investigated using a modified LAMMA 1000 reflecting time-of-flight (TOF) mass spectrometer. Whereas fragmentation of covalent bonds prior to ion acceleration (i.e., within several ns after the laser pulse) in general is not observed using the matrix technique, extensive fragmentation on a longer time scale can be studied in our instrument. The high mass resolution (MIAM= 1200-1800 for insulin and peptides) permits the investigation of even small mass losses from parent molecular ions (occurring in the first section of the field-free drift region) by measuring flight time differences of daughter ions acquired during passage through a two-stage reflectron. The kind and extent of this metastable decay has been found to depend strongly on the substance under investigation. Typical fragmentations are loss of ammonia and parts of the amino acid side-chains. The large abundances of peaks due to such metastable fragmentation, observed for most of the peptides and proteins investigated, may, at least in part, explain peak broadening (and, hence, poor mass resolution) typical in matrix-assisted laser-desorption TOF mass spectra.One of the main goals in matrix-assisted laser desorption of high-mass biopolymers, a technique first described by Karas and Hillenkamp, ' is the achievement of high mass resolution and high mass accuracy for the full mass range (up to ca 300 000 Da) accessible to date. For several proteins 'having molecular weights below 30 000 Da, Beavis and Chait reported a mass resolving power, inlAm, of 300-500 leading to an accuracy in mass measurement of about ?0.01% by means of internal calibrants.' However, mass spectra of proteins in the very high mass region as reported in the literature have degraded mass resolutions, reaching values of not higher than ca 50 in the range above ca 100 000 Da. Since, in this mass range, the detection of the ions is governed by processes of secondary-ion rather than secondary-electron formation at the instrument's target surfaces (such as a conversion dynode, an ion detector or field grids3-'), spread in flight times of these secondary species has been suspected to cause substantial peak b r~a d e n i n g .~ However, even under instrumental conditions that carefully avoid such secondary-ion formation at or near the detector, mass resolution remains much lower than expected. We report here on another mechanism that contributes to peak broadening, resulting from the lack of stability of high-mass ions during their flight through the time-of-flight (TOF) mass spectrometer. It is shown that a substantial number of ions decay after acceleration and prior to detection, even under threshold conditions of laser irradiation. This leads to peak broadening by two mechanisms: (i) flight-time differences between neutrals, parent ions and fragment ions resulting from their passage through electric fields (e.g., ion lenses, fringe fields in front of channel-plate detectors, ion converters etc....

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.

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The determination of the age of an ink entry from a questioned document is often a major problem and a controversial issue in forensic sciences. Therefore, it is important to understand the aging process of the different components found in ink. The aim of this work is to characterize the degradation processes of methyl violet and ethyl violet, two typical ballpoint dyes by using laser desorption/ionization (LDI) and matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry (MS), and to evaluate the possible application of the method to forensic examination of documents. The mass spectrometric methods were first tested and were found to be adequate for the purpose of this work. Moreover, it is possible to analyze the dye from a stroke directly from the paper (LDI-MS), so the sample preparation is minimized. The degradation of the dyes methyl violet and ethyl violet in strokes from a ballpoint pen was studied under laboratory conditions influenced by different factors such as light, wavelength of light, heat, and humidity. Then, strokes from the same ballpoint were aged naturally in the dark or under the influence of light over one year and then analyzed. The results show that the degradation of these dyes strongly depends on light fluence. Humidity also increases degradation, which can be explained by the basicity of the paper. The influence of heat on the degradation process was found to be rather weak. It was also observed that the dyes from the ink strokes did not show significant degradation after one year of storage in the dark. In conclusion, the storage conditions of a questioned document and the initial composition of the dyes in the ink have to be known for correct interpretation of the age of an ink entry. Measurements over longer periods of time are necessary to follow the degradation of dyes exempt from light exposure. LDI was found adequate and very useful for the analysis of ballpoint dyes directly from paper without further pretreatment. n the field of forensic examination of questioned documents, the legitimacy of an ink entry is often an essential question, and the possibility of determining the age of an ink stroke would definitely help to resolve this problem. Ballpoint pens are very common scriptural instruments, the inks of which contain equivalent amounts of dyes, solvents, and resins. After deposition on paper, the ink composition begins to change qualitatively and quantitatively: the resins polymerize, the solvents evaporate, and the dyes fade. Throughout the years, many different methods were developed to measure the changes occurring in the ink with time [1][2][3][4][5][6][7]: decrease of extractability of ink through hardening of the resins [8 -13, 16], disappearance of solvents [14 -20], and degradation of dyes [18,[21][22][23][24][25][26][27][28][29]. The analysis of the latter compounds involve techniques such as microspectrophotometry [21], HPLC analysis [25,26], or recently mass spectrometry [18, 22-24, 27, 28]. Laser desorption/ionization mass spectrometry (LDI-MS) i...