Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucoselimited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that currently affects 36 million people worldwide with no effective treatment available. Development of AD follows a distinctive pattern in the brain and is poorly modelled in animals. Therefore, it is vital to widen the spatial scope of the study of AD and prioritise the study of human brains. Here we show that functionally distinct human brain regions display varying and region-specific changes in protein expression. These changes provide insights into the progression of disease, novel AD-related pathways, the presence of a gradient of protein expression change from less to more affected regions and a possibly protective protein expression profile in the cerebellum. This spatial proteomics analysis provides a framework which can underpin current research and open new avenues to enhance molecular understanding of AD pathophysiology, provide new targets for intervention and broaden the conceptual frameworks for future AD research.

Tandem mass spectrometry (MS/MS) of peptides plays a key role in the field of proteomics, and an understanding of the fragmentation mechanisms involved is vital for data interpretation. Not all the fragment ions observed by low-energy collision-induced dissociation of protonated peptides are readily explained by the generally accepted structures for a-and b-ions. The possibility of a macrocyclic structure for b-type ions has been recently proposed. In this study, we have undertaken investigations of linear protonated YAGFL-NH 2 , N-acetylated-YAGFL-NH 2 , and cyclo-(YAGFL) peptides and their fragments using a combination of ion mobility (IM) separation and mass spectrometry. The use of IM in this work both gives insight into relative structural forms of the ion species and crucial separation of isobaric species. Our study provides compelling evidence for the formation of a stable macrocyclic structure for the b 5 ion generated by fragmentation of protonated linear YAGFL-NH 2 . Additionally we demonstrate that the a 4 ion fragment of protonated YAGFL-NH 2 has at least two structures; one of which is attributable to a macrocyclic structure on the basis of its subsequent fragmentation. More generally, this work emphasizes the value of combined IM-MS/MS in probing the detailed fragmentation mechanisms of peptide ions, and illustrates the use of combined ion mobility/ collisional activation/mass spectrometry analysis in achieving an effective enhancement of the resolution of the mobility separator. . Accordingly, it is important to understand the gas-phase ion chemistry that underpins the MS/MS of peptide ions in order to optimize the application of the technique. The structures of the N-terminal a-and b-ions formed from the fragmentation of collisionally activated protonated peptides in the gas phase [2] have been a topic of investigation and discussion for several years. The b-ions have generally been considered to consist of a linear peptide chain terminating in a cyclic oxazolone structure (Scheme 1a) [3]; subsequent formation of a-ions by loss of CO has been presumed to follow opening of the oxazolone ring. These hypotheses alone, however, do not readily explain all the fragment ions observed in low-energy collision-induced dissociation experiments. Yagüe et al.[4] proposed rearrangement of b-ions to give macrocyclic intermediate structures that could fragment further to yield products in more complete agreement with observed data. In earlier work, Tang and Boyd [5] reported evidence of an interaction between the primary amine groups of lysine or ornithine residues with the C-terminus of peptide ions. Recently, Harrison et al.[6] used both computational studies and comparison of the breakdown graphs of the b 5 ion (produced by fragmentation of protonated YAGFL-NH 2 ) and the protonated cyclo-(YAGFL) peptide analogue to provide strong evidence for a cyclic structure for the b 5 -ion moiety (Scheme 1b). Current ideas [4,[7][8][9] suggest a mixture of possible linear and cyclic structures for a-ions.To gain further...