This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 ␣-amino acids, ␤-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither ␤-, ␥-, nor ␦-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong ince their introduction in the late 1980s, the soft-ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization (MALDI), have became the major ionization methods for the analysis of biological molecules. While analysis of large biopolymers is successfully performed by both techniques, analysis of low molecular weight (LMW) molecules such as amino acids, pharmaceuticals, hormones, etc. is predominantly performed by ESI mass spectrometry with quadrupole mass analyzers. The major reasons not to use MALDI have been the abundance of matrix ions in the low mass region (100 to 600 u) and the lack of reasonable precursor selection in a MALDI tandem instrument. Also, until recently, MALDI was not considered to be a technique capable of providing robust quantitative data. Current developments in MALDI hardware [1,2] and increased attention paid to sample preparation have changed the situation. A number of recent publications describe application of MALDI TOF for quantitative studies [3][4][5][6][7][8], including favorable comparisons of MALDI TOF with the more traditional ESI LC/MS quantitation schemes [9 -11]. When installed on QqQ instruments, a MALDI source demonstrated good comparability with an ESI source on the same instrument in quantitative analysis of LMW compounds [12].Amino acid analysis (AAA) is one of the areas where ESI LC/MS/MS was successfully applied. ESI LC/ MS/MS has become a standard analytical tool in the analysis of low molecular weight physiological or therapeutic molecules of interest [13][14][15][16].We decided to examine if the combination of highenergy collision and prompt ion detection characteristic for MALDI TOF MS/MS could be applied for AAA. The proposed application of MALDI TOF for AAA contains some advantages o...

Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver. These results, and contemporary work by others, indicate that protein covalent binding is not totally random in cells. Moreover, as protein targets for other protoxins were identified, little commonality of target proteins became apparent. In the present work, we used two-dimensional gel electrophoresis to separate liver cytosolic proteins from rats treated with 14C-bromobenzene; 110 of the 836 observed spots contained measurable radioactivity that varied over a 600-fold range of adduct density. Of these 110 spots, in-gel digestion coupled with mass spectrometry identified apparently single proteins in 57 spots. A few other spots clearly contained more than one identifiable protein, and in several cases, the same protein was identified in several spots having different apparent molecular masses and/or pI. Altogether, 33 unique new protein targets for bromobenzene metabolites were identified and compared to those known for acetaminophen, naphthalene, butylated hydroxytoluene, benzene, thiobenzamide, and halothane via a target protein database available at http://tpdb.medchem.ku.edu:8080/protein_database/. With increasing numbers of target proteins becoming known, more commonality in targeting by reactive metabolites from diverse chemical agents may be seen. Such commonality may help to separate toxicologically significant covalent binding events from a background of covalent binding that is toxicologically inconsequential.

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as 'lipid rafts' contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed agedependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p < 0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain.

Here, we describe two different amino acid analysis protocols based on the application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates (Alterman et al. Anal Biochem 335: 184-191, 2004). Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM. Limit of quantitation varied from 0.03 μM for arginine to 3.7 μM for histidine and homocysteine. This method has one inherent limitation: the analysis of isomeric and isobaric amino acids. To solve this problem, a second protocol based on the use of MALDI TOF/TOF MS/MS for qualitative analysis of amino and organic acids was developed. This technique is capable of distinguishing isobaric and isomeric compounds (Gogichayeva et al. J Am Soc Mass Spectrom 18: 279-284, 2007). Both methods do not require amino acid derivatization or chromatographic separation, and the data acquisition time is decreased to several seconds for a single sample.

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