Metastable decay of peptides and proteins in matrix‐assisted laser‐desorption mass spectrometry

Abstract

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....

MALDI In-Source Decay, from Sequencing to Imaging

“…Fragmentations occur by collision induced dissociation (CID), by electrons as in electron capture dissociation (ECD) [6,12,13], or by electron transfer dissociation (ETD) [14]. Until recently, TDS was not intensively used with MALDI due to the fact that the produced ions are singly charged and MS/MS techniques available on MALDI mass spectrometers are efficient only for peptides up to m/z 3,500 [12]. Indeed, PSD and CID fragmentation processes rely on a slow increase of the ion internal energy by multiple collisions with gas molecules until the lower energy bonds are dissociated (mainly labile or peptide bonds).…”

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

“…M etastable decomposition occurs universally during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses of molecules having labile bonds. Although this phenomenon can be used for obtaining analyte structural information for small peptides, oligosaccharides, and some glycolipids [1][2][3][4][5] for larger or more fragile molecules, it can limit the mass accuracy and resolution and complicate interpretation of the spectra of heterogeneous samples. Thus, it is important to develop methods that allow control of the extent of metastable decay.…”

Proteome imaging: A closer look at life's organization

“…This was the conception of direct molecular imaging of biological tissue with MS. When a few years later the first MALDI results were reported demonstrating the direct desorption and ionization of intact proteins it was obvious that the LAMMA approach would soon be applied to surfaces that were covered with matrices [50]. In the next section the current status of MS-based proteome imaging technology will be reviewed and the microprobe and microscope approach for MS-based biomolecular imaging compared.…”