Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried - from expression host - six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Alterations in the Salivary Proteome and N-Glycome of Sjögren's Syndrome Patients.

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of ß2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection.

UNLABELLED: Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity. IMPORTANCE: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen.

In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses.

OBJECTIVE: To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients. METHODS: Mass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients. RESULTS: We find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood-brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related. INTERPRETATION: Our data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier.

Aberrant hetero-disulfide bond formation by the hypertriglyceridemia-associated p.Gly185Cys APOA5 variant (rs2075291).

OBJECTIVE: Apolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia. APPROACH AND RESULTS: Adeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others. CONCLUSIONS: Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.

Interaction of fibronectin with semen amyloids synergistically enhances HIV infection.

Semen harbors amyloids that enhance human immunodeficiency virus type 1 (HIV-1) infection. We set out to identify factors that bind these amyloids and to determine whether these factors modulate amyloid-mediated HIV-enhancing activity. Using biochemical and mass spectrometric approaches, we identified fibronectin as a consistent interaction partner. Although monomeric fibronectin did not enhance HIV infection, it synergistically increased the infectivity enhancement activity of the amyloids. Depletion of fibronectin decreased the enhancing activity of semen, suggesting that interfering with the binding interface between fibronectin and the amyloids could be an approach to developing a novel class of microbicides targeting the viral-enhancing activity of semen.

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions.

BACKGROUND: Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated. METHODS: Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS. RESULTS: It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents. CONCLUSION: These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization. GENERAL SIGNIFICANCE: This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 µm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers.

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

Survey of large protein complexes in D. vulgaris reveals great structural diversity.

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximately 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximately 10 times greater than that of previous "proteomic" screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

Evidence-based annotation of transcripts and proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

We used high-resolution tiling microarrays and 5' RNA sequencing to identify transcripts in Desulfovibrio vulgaris Hildenborough, a model sulfate-reducing bacterium. We identified the first nucleotide position for 1,124 transcripts, including 54 proteins with leaderless transcripts and another 72 genes for which a major transcript initiates within the upstream protein-coding gene, which confounds measurements of the upstream gene's expression. Sequence analysis of these promoters showed that D. vulgaris prefers -10 and -35 boxes different from those preferred by Escherichia coli. A total of 549 transcripts ended at intrinsic (rho-independent) terminators, but most of the other transcripts seemed to have variable ends. We found low-level antisense expression of most genes, and the 5' ends of these transcripts mapped to promoter-like sequences. Because antisense expression was reduced for highly expressed genes, we suspect that elongation of nonspecific antisense transcripts is suppressed by transcription of the sense strand. Finally, we combined the transcript results with comparative analysis and proteomics data to make 505 revisions to the original annotation of 3,531 proteins: we removed 255 (7.5%) proteins, changed 123 (3.6%) start codons, and added 127 (3.7%) proteins that had been missed. Tiling data had higher coverage than shotgun proteomics and hence led to most of the corrections, but many errors probably remain. Our data are available at http://genomics.lbl.gov/supplemental/DvHtranscripts2011/.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

Automated iterative MS/MS acquisition: a tool for improving efficiency of protein identification using a LC-MALDI MS workflow.

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ∼20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma.

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

The sodium-hydrogen exchanger NHE1 is an Akt substrate necessary for actin filament reorganization by growth factors.

The kinase Akt mediates signals from growth factor receptors for increased cell proliferation, survival, and migration, which contribute to the positive effects of Akt in cancer progression. Substrates are generally inhibited when phosphorylated by Akt; however, we show phosphorylation of the plasma membrane sodium-hydrogen exchanger NHE1 by Akt increases exchanger activity (H(+) efflux). Our data fulfill criteria for NHE1 being a bona fide Akt substrate, including direct phosphorylation in vitro, using mass spectrometry and Akt phospho-substrate antibodies to identify Ser(648) as the Akt phosphorylation site and loss of increased exchanger phosphorylation and activity by insulin and platelet-derived growth factor in fibroblasts expressing a mutant NHE1-S648A. How Akt induces actin cytoskeleton remodeling to promote cell migration and tumor cell metastasis is unclear, but disassembly of actin stress fibers by platelet-derived growth factor and insulin and increased proliferation in growth medium are inhibited in fibroblasts expressing NHE1-S648A. We predict that other functions shared by Akt and NHE1, including cell growth and survival, might be regulated by increased H(+) efflux.

Acid-catalyzed oxygen-18 labeling of peptides.

In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.

Rapid alterations in cortical protein profiles underlie spontaneous sleep and wake bouts.

Existing data indicate that sleep-wakefulness is an essential behavior. The biological function(s) of sleep, however, remains unknown, due, in part, to the lack of information available at the intracellular level. Preliminary microarray analyses show that changes in behavioral state influence regional mRNA profiles; however, the impact of sleep on protein signatures is virtually unexplored. In these studies, cortical protein profiles were examined after timed bouts of spontaneous sleep-wakefulness. Within minutes of each behavioral state examined, a small number of spots showing unique expression were detected. Mass spectroscopy analyses of sleep- and wake-related spots identified proteins associated with multiple functional categories. Two sleep-associated proteins were further validated using a sleep deprivation paradigm. We found preliminary evidence for two different post-transcriptional mechanisms-one (GAPDH) in which the amount of protein was increased in the recovery sleep following prolonged waking, while the other (actin) suggested that post-translational modifications may underlie sleep. The similarities between the effects of sleep on both protein and mRNA profiles indicate that dynamic intracellular changes underlie sleep-wake states and are consistent with roles for sleep in multiple biological functions.