Background:Researchers typically search for disease markers using a "targeted" approach in which a hypothesis about the disease mechanism is tested and experimental results either confirm or disprove the involvement of a particular gene or protein in the disease. Recently, there has been interest in developing disease diagnostics based on unbiased quantification of differences in global patterns of protein and peptide masses, typically in blood from individuals with and without disease. We combined a suite of methods and technologies, including novel sample preparation based on carrier-protein capture and biomarker enrichment, highresolution mass spectrometry, a unique cohort of wellcharacterized persons with and without Alzheimer disease (AD), and powerful bioinformatic analysis, that add statistical and procedural robustness to biomarker discovery from blood. Methods: Carrier-protein-bound peptides were isolated from serum samples by affinity chromatography, and peptide mass spectra were acquired by a matrixassisted laser desorption/ionization (MALDI) orthogo-
Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)–mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated forBacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.
Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides. 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 [1-5] 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. In addition to the pressure of the background gas and extraction field strength in the MALDI technique, the major factors affecting metastable fragmentation are laser wavelength/pulse width and the choice of the MALDI matrix [6,7]. In addition, the presence of electrons may play a role in analyte fragmentation and may cause suppression of multiply charged ions in a spectrum [8,9].Control of the metastable fragmentation in MALDI by increasing the pressure in the ionization source and reduction of the ions internal energy was first introduced by Loboda et al. on orthogonal time-of-flight (oTOF) instruments [10] and later extended to Fourier transform mass spectrometry (FTMS) [11] and to the operation at atmospheric pressure on various types of instruments [12]. These methods have since been used for stabilization of glycolipids [13,14] and peptides and proteins [11,15]. The term vibrational cooling (VC) MALDI recently has been introduced to describe desorption in the pressure range where cooling of the excess bond energy is achieved [12]. Stabilization of the labile bonds under these conditions allows for control of the extent of fragmentation, which is particularly
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