O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength. Molecular & Cellular Proteomics 7:684 -696, 2008.Synaptic transmission between neurons in the central nervous system involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels in the surface membrane of postsynaptic neurons. These neurotransmitter receptors exist as part of a highly organized protein complex known as the postsynaptic density (PSD).1 In addition to neurotransmitter receptors, the PSD is composed of proteins of diverse function (1, 2). These functional groups include scaffolding molecules, kinases and phosphatases, G proteins and their effectors, and adhesion proteins.The coordinated functioning of the different PSD components regulates in part the strength of signaling between the pre-and postsynaptic neurons. At the molecular level, this regulation can be achieved by alterations in protein localization (3-5), reversible post-translational modifications (e.g. phosphorylation (6, 7) and O-GlcNAc glycosylation (8)), and changes in protein levels via local synthesis and degradation (9). Integration of these processes may be the basis of long term changes in synaptic efficacy thought to underlie higher cognitive processes such as learning and memory (10).Proteomics approaches aimed at identifying synaptic proteins (11-16) and their post-translational modificat...

Mass spectrometric analyses of protein digests produce large numbers of fragmentation spectra that are not identified by routine database searching strategies. Some of these spectra could be identified by development of improved search engines. However, many of these spectra represent fragmentation of peptide components bearing modifications that are not routinely considered in database searches. Here we present new software within Protein Prospector that allows comprehensive analysis of data sets by analyzing the data at increasing levels of depth. Analysis of published data sets is presented to illustrate that the software is not biased to any instrument types. The results show that these data sets contain many modified peptides. As well as searching for known modification types, Protein Prospector permits the detection and identification of unexpected or novel modifications by searching for any mass shift within a user-specified mass range to any chosen amino acid(s). Several modifications never previously reported in proteomics data were identified in these standard data sets using this mass modification searching approach. Molecular & Cellular Proteomics 7:2386 -2398, 2008.

Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k cat ∕K M ) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.apoptosis | caspase | enzymology | mass spectrometry | selected reaction monitoring A poptosis is a form of programmed cell death that serves to eliminate unnecessary, infected, or tumorigenic cells from eukaryotic organisms. While many intrinsic and extrinsic stimuli can initiate apoptosis, these ultimately converge on the activation of a related family of aspartate-specific cysteine proteases, the caspases, that execute widespread proteolysis and induce noninflammatory death (1). We and others have surveyed N termini that occur in apoptotic cells and collectively reported more than 1,000 caspase-derived cleavages (2-5). This explosion of proteomic data has defined a vast array of caspase substrates proteolyzed during apoptosis. While these data identify caspase targets, and in some cases the sites of proteolysis, they fail to reveal the relative rates of cleavage, a parameter necessary to establish the order of proteolytic events and their importance in extracts and intact cells.The recent application of selected reaction monitoring (SRM) methods, traditionally used for metabolite identification, to proteomic studies has enabled the simultaneous label-free quantification of hundreds of peptides (6, 7). Our development of a N-terminal enrichment platform (3) is ideally suited to the application of SRM to apoptotic proteolysis. Using this platfor...

SummaryIt is well established that protein sequence determination may be achieved by mass spectrometric analysis of protonated tryptic peptides subjected to collisional activation. When separated by nanoflow HPLC, a high percentage of peptides from complex mixtures of proteins can usually be identified.Recently alternative, radical-driven fragmentation approaches of electron capture dissociation and the more common electron transfer dissociation (ETD) have been introduced and made widely available. In order to utilize these techniques in large scale proteomics studies it is important to characterize the performance of these fragmentation processes on peptides formed by a range of enzymatic cleavages.In this study we present a statistical analysis of the ion types that are observed from peptides produced by different enzymes, and highlight the different characteristics of ETD spectra of doubly-charged precursors in comparison to precursors of higher charge states.

Peripheral nerve injuries elicit a cascade of axonal responses that are required for a successful regenerative response. The lesioned axons must signal retrogradely to their cell bodies to activate intrinsic neurite outgrowth mechanisms (1-3) and then overcome physical barriers and inhibitory cues in the extracellular environment to achieve functional regeneration (4, 5). The first indications of a breach in axonal integrity upon injury are most likely abnormal generation of action potentials and/or waves of calcium propagating from the lesion site toward the intact portions of the cell (6, 7). At a later stage, signals carried by motor-driven transport systems start to affect the cell body. This phase includes both an interruption of the normal supply of retrogradely transported molecules such as trophic factor signals (8) and arrival of new signals elicited at the injury site (3, 9). The latter interact with a variety of dynein-associated carriers including importins (10, 11) and kinase family scaffolds (12, 13).In mammalian neurons, the distances between axonal lesion sites and the nucleus can reach many centimeters (up to 1 m in humans); hence, retrograde signaling events within the first few hours after injury must be independent of new transcription in the cell body. Proteolysis, local protein synthesis, and post-translational signaling modifications such as phosphorylation have all been implicated in generation of the retrograde signaling ensemble (1). This prominent role for post-transcriptional processes suggests that comprehensive characterization of retrograde signaling will require proteomics approaches. In a previous study we used two-dimensional PAGE and mass spectrometry to analyze retrogradely concentrated axoplasm from injured mollusc nerve, identifying a vesicular ensemble blocked by the lesion and an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament (14,15). Follow-up studies in rodent sciatic nerve showed that the mammalian intermediate filament vimentin is produced by local translation of axonal mRNA upon axonal injury and then undergoes calpain-mediated proteolysis, generating a cleavage product that interacts with importins bound to dynein and enables protected retrograde transport of phosphorylated forms of the mitogen-activated protein kinases Erk1 and Erk2 (16,17). Here, we extend our efforts to determine the components of the retrograde injury signaling ensemble in lesioned nerve by using LC-MS/MS coupled with iTRAQ TM1 labeling to directly analyze mammalian axoplasm samples after nerve injury. The analyses reveal extensive changes in both anterograde and Research

Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (lESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during lES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the a subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.

This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopep-tides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that “old” proteomic data may yield significant new information when re-interrogated with new, improved tools.