No abstract

Systematic investigations of particle suspensions for the laser desorption/ionization of peptides and proteins are presented. The performance and suitability for time-of-flight mass spectrometry of different particle materials and sizes, suspended in a variety of different liquids, are described. Performance characteristics such as accessible mass range, achievable mass resolution, analytical sensitivity, and fragmentation are reported. For the desorption of peptides and small proteins, nanoparticle suspensions in glycerol were found to perform comparably to UV-MALDI-MS with common "chemical" matrixes. For proteins in the mass range of ∼12-30 kDa, mass resolution and analytical sensitivity decrease sizeably; for proteins with masses in excess of ∼30 kDa, no spectra could be recorded with any of the tested particle/liquid combinations. The results were found to be largely independent of the laser wavelength in the range from the near-UV to the near-IR because of the strong particle absorption throughout this wavelength range. Ions are shown to originate predominantly from analyte molecules adsorbed at the particle surface. Nanoparticles with a diameter of a few nanometers were found to be superior to microparticles of ∼1 μm diameter or above. Thermodynamic modeling suggests that this different behavior is caused by the different achieved peak temperatures of the two particle sizes.

Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.