A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated. (J Am Soc Mass Spectrom 2002, 13, 784 -791)

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High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.

A detailed description of a linear ion trap time-of-flight (TOF) mass spectrometer system, capable of sequential mass spectrometry (MS(n)), is given. Many improvements have been incorporated since the initial description of this system (Rapid Commun. Mass Spectrom. 1998; 12: 1463-1474). The pressure in the trap has been lowered from 7.0 to 1.8 mTorr, resulting in an increase in the mass resolution of ion excitation from 75 to 240. Use of the system for MS(3) is demonstrated. Dipole excitation of the n = 1 harmonic, instead of the n = 0 fundamental frequency of ion motion, is shown to have a higher frequency resolution, f/Deltaf, but lower mass resolution, m/Deltam. Both experiments and modeling demonstrate that at the lower pressure there is less collisional cooling of ions in the axial and radial directions of the trap. The efficiency of trapping is shown to be nearly 100% for periods up to 5 s. The demonstrated mass range for mass analysis has been extended to greater than m/z 16 250. To avoid the formation of adduct ions when trapping protein ions for extended times requires ultra-high vacuum cleanliness conditions, even though the trap operates in the mTorr-pressure range. Upgrading the TOF to a reflectron with higher quality ion optics results in an increase in the mass resolution of the TOF mass spectrometer to about 5000 at m/z 750.