A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In the current study, we show that transgenic mice modeling Alzheimer disease-PDAPP mice with Swedish and Indiana mutations in human amyloid precursor protein (APP)-develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3-nitrotyrosine) and lipid peroxidation (indexed by protein-bound 4-hydroxy-2-nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid β-peptide (Aβ), since all indices of oxidative damage in brain were completely prevented in genetically and age-matched PDAPP mice with a M631L mutation in APP. No significant differences in levels of APP, Aβ(1-42), Aβ (1-40), or the ratio Aβ(1-42)/Aβ(1-40) were found, suggesting that the loss of oxidative stress in vivo in brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Aβ peptide. However, a marked reduction in Aβ-immunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of non-plaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Aβ sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutation-PDAPP(D664A)-showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Aβ residue Met35 for oxidative stress in brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities.

Liquid chromatographies coupled to mass spectrometry and database analysis techniques are used to carry out a large-scale proteome characterization for a Drosophila model of Parkinson's disease. Semi-quantitative analysis is performed on A30P α-synuclein expressing transgenic Drosophila and a control lacking the gene at pre-symptomatic, early and advanced disease stages. Changes in gene expression at the level of the proteome are compared with changes reported from published transcriptome measurements. A summary of the comparison indicates that ~44% of transcripts that show changes can also be observed as proteins. However, the patterns of change in protein expression vary substantially compared with the patterns of change observed for corresponding transcripts. In addition, the expression changes of many genes are observed for only transcripts or proteins. Proteome measurements provide evidence for dysregulation of a group of proteins associated with the actin cytoskeleton and mitochondrion at pre-symptomatic and early disease stages that may presage the development of later symptoms. Overall, the proteome measurements provide a view of gene expression that is highly complementary to the insights obtained from the transcriptome.

A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T ␣-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 (ϳ71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T ␣-synuclein

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.