Search

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key

Mycobacterium avium secretes iron-binding siderophores called exochelins. The exochelins from M. avium have previously been reported to have unsaturated side chains that terminate in carboxylic acid. In contrast, our data show the side chains to be both saturated and unsaturated and to terminate with either a carboxylate or methyl ester.

Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores called exochelins. Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium. In this paper, we describe the purification of exochelins of M. tuberculosis and their characterization by mass spectrometry. Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state. The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain. These series further subdivide into serine- or threonine-containing species. The virulent M. tuberculosis Erdman strain and the avirulent M. tuberculosis H37Ra strain produce a similar set of exochelins. Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins. However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester. These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

Advances in proteomic assay technologies have significantly increased coverage and throughput, enabling recent increases in the number of large-scale population-based proteomic studies of human plasma and serum. Improvements in multiplexed protein assays have facilitated the quantification of thousands of proteins over a large dynamic range, a key requirement for detecting the lowest-ranging, and potentially the most disease-relevant, blood-circulating proteins. In this perspective, we examine how populational proteomic datasets in conjunction with other concurrent omic measures can be leveraged to better understand the genomic and non-genomic correlates of the soluble proteome, constructing biomarker panels for disease prediction, among others. Mass spectrometry workflows are discussed as they are becoming increasingly competitive with affinity-based array platforms in terms of speed, cost, and proteome coverage due to advances in both instrumentation and workflows. Despite much success, there remain considerable challenges such as orthogonal validation and absolute quantification. We also highlight emergent challenges associated with study design, analytical considerations, and data integration as population-scale studies are run in batches and may involve longitudinal samples collated over many years. Lastly, we take a look at the future of what the nascent next-generation proteomic technologies might provide to the analysis of large sets of blood samples, as well as the difficulties in designing large-scale studies that will likely require participation from multiple and complex funding sources and where data sharing, study designs, and financing must be solved., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BBS is an employee of Bristol Myers Squibb and former employee of Biogen., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 μg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

The Pharma Proteomics Project is a precompetitive biopharmaceutical consortium characterizing the plasma proteomic profiles of 54,219 UK Biobank participants. Here we provide a detailed summary of this initiative, including technical and biological validations, insights into proteomic disease signatures, and prediction modelling for various demographic and health indicators. We present comprehensive protein quantitative trait locus (pQTL) mapping of 2,923 proteins that identifies 14,287 primary genetic associations, of which 81% are previously undescribed, alongside ancestry-specific pQTL mapping in non-European individuals. The study provides an updated characterization of the genetic architecture of the plasma proteome, contextualized with projected pQTL discovery rates as sample sizes and proteomic assay coverages increase over time. We offer extensive insights into trans pQTLs across multiple biological domains, highlight genetic influences on ligand-receptor interactions and pathway perturbations across a diverse collection of cytokines and complement networks, and illustrate long-range epistatic effects of ABO blood group and FUT2 secretor status on proteins with gastrointestinal tissue-enriched expression. We demonstrate the utility of these data for drug discovery by extending the genetic proxied effects of protein targets, such as PCSK9, on additional endpoints, and disentangle specific genes and proteins perturbed at loci associated with COVID-19 susceptibility. This public-private partnership provides the scientific community with an open-access proteomics resource of considerable breadth and depth to help to elucidate the biological mechanisms underlying proteo-genomic discoveries and accelerate the development of biomarkers, predictive models and therapeutics 1 ., (© 2023. The Author(s).)

Anesthesia is often used in preclinical imaging studies that incorporate mouse or rat models. However, multiple reports indicate that anesthesia has significant physiological impacts. Thus, there has been great interest in performing imaging studies in awake, unanesthetized animals to obtain accurate results without the confounding physiological effects of anesthesia. Here, we describe a newly designed mouse holder that is interfaceable with existing MRI systems and enables awake in vivo mouse imaging. This holder significantly reduces head movement of the awake animal compared to previously designed holders and allows for the acquisition of improved anatomical images. In addition to applications in anatomical T 2 -weighted magnetic resonance imaging (MRI), we also describe applications in acquiring 31 P spectra, manganese-enhanced magnetic resonance imaging (MEMRI) transport rates and resting-state functional magnetic resonance imaging (rs-fMRI) in awake animals and describe a successful conditioning paradigm for awake imaging. These data demonstrate significant differences in 31 P spectra, MEMRI transport rates, and rs-fMRI connectivity between anesthetized and awake animals, emphasizing the importance of performing functional studies in unanesthetized animals. Furthermore, these studies demonstrate that the mouse holder presented here is easy to construct and use, compatible with standard Bruker systems for mouse imaging, and provides rigorous results in awake mice.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability.", Competing Interests: Conflict of interest The authors declare the following competing financial interest(s): N. B., A. M., H. W., T. M. A., S.-C. O., Y. Z., X. S., D. N. B., H. R., B. W. G., B. M. R., and S. C. H. are employees and stockholders of Amgen. K. M. R., D. L. D., and M. U. are employees and stockholders of Promega Corporation., (Published by Elsevier Inc.)

Thorough characterization of protein therapeutics is often challenging due to the heterogeneity arising from primary sequence variants, post-translational modifications, proteolytic clipping, or incomplete processing of the signal peptide. Modern mass spectrometry (MS) techniques are now routinely used to characterize such heterogeneous protein populations. Here, we present an LC-MS/MS method using ( N -succinimidyloxycarbonylmethyl)-tris (2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to label any free N-terminal α-amines to rapidly and selectively identify proteolytic clipping events. Electron transfer dissociation (ETD) fragmentation of these chemically tagged peptides generates two unique TMPP product ions, TMPP + and TMPP-Ac-NH 2 /c0. The presence of these signature ions following ETD is used to trigger subsequent collisional induced dissociation (CID) fragmentation of the precursor ion. This results in a small subset of CID tandem MS spectra that are used in a customized database search. Using a purified fusion monoclonal antibody (mAb) as an example, we demonstrate how TMPP labeling followed by ETD product ion triggered CID fragmentation is used to accurately identify two undesired clipping sites.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE). The molecular mechanisms leading to RPE dysfunction in response to ROS are unclear. Here, human stem cell-derived RPE samples were stressed with ROS for 1 or 3 weeks, and both intracellular and secreted proteomes were quantified by mass spectrometry. ROS increased glycolytic proteins but decreased mitochondrial complex I subunits, as well as membrane proteins required for endocytosis. RPE secreted over 1,000 proteins, many of which changed significantly due to ROS. Notably, secreted APOE is decreased 4-fold, and urotensin-II, the strongest known vasoconstrictor, doubled. Furthermore, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, our results paint a detailed molecular picture of the retinal stress response in space and time.

Dietary sugars, fructose and glucose, promote hepatic de novo lipogenesis and modify the effects of a high-fat diet (HFD) on the development of insulin resistance. Here, we show that fructose and glucose supplementation of an HFD exert divergent effects on hepatic mitochondrial function and fatty acid oxidation. This is mediated via three different nodes of regulation, including differential effects on malonyl-CoA levels, effects on mitochondrial size/protein abundance, and acetylation of mitochondrial proteins. HFD- and HFD plus fructose-fed mice have decreased CTP1a activity, the rate-limiting enzyme of fatty acid oxidation, whereas knockdown of fructose metabolism increases CPT1a and its acylcarnitine products. Furthermore, fructose-supplemented HFD leads to increased acetylation of ACADL and CPT1a, which is associated with decreased fat metabolism. In summary, dietary fructose, but not glucose, supplementation of HFD impairs mitochondrial size, function, and protein acetylation, resulting in decreased fatty acid oxidation and development of metabolic dysregulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Dietary macronutrient composition alters metabolism through several mechanisms, including post-translational modification (PTM) of proteins. To connect diet and molecular changes, here we performed short- and long-term feeding of mice with standard chow diet (SCD) and high-fat diet (HFD), with or without glucose or fructose supplementation, and quantified liver metabolites, 861 proteins, and 1,815 protein level-corrected mitochondrial acetylation and succinylation sites. Nearly half the acylation sites were altered by at least one diet; nutrient-specific changes in protein acylation sometimes encompass entire pathways. Although acetyl-CoA is an intermediate in both sugar and fat metabolism, acetyl-CoA had a dichotomous fate depending on its source; chronic feeding of dietary sugars induced protein hyperacetylation, whereas the same duration of HFD did not. Instead, HFD resulted in citrate accumulation, anaplerotic metabolism of amino acids, and protein hypo-succinylation. Together, our results demonstrate novel connections between dietary macronutrients, protein post-translational modifications, and regulation of fuel selection in liver., Competing Interests: The authors have declared that no competing interests exist.

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label-free proteomic workflow-"one-pot" PTM enrichment-that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label-free, data-independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one-pot method are identified and quantified and peak areas are shown to be highly correlated between the one-pot and traditional single-PTM enrichments. The 'one-pot' method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue., (© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Post-translational modification of lysine residues via reversible acylation occurs on proteins from diverse pathways, functions, and organisms. While nuclear protein acylation reflects the competing activities of enzymatic acyltransferases and deacylases, mitochondrial acylation appears to be driven mostly via a non-enzymatic mechanism. Three protein deacylases, SIRT3, SIRT4, and SIRT5, reside in the mitochondria and remove these modifications from targeted proteins in an NAD + -dependent manner. Recent proteomic surveys of mitochondrial protein acylation have identified the sites of protein acetylation, succinylation, glutarylation, and malonylation and their regulation by SIRT3 and SIRT5. Here, we review recent advances in this rapidly moving field, their biological significance, and their implications for mitochondrial function, metabolic regulation, and disease pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transcriptional regulation is the key to ensuring that proteins are expressed at the proper time and the proper amount. In Escherichia coli, the transcription factor cAMP receptor protein (CRP) is responsible for much of this regulation. Questions remain, however, regarding the regulation of CRP activity itself. Here, we demonstrate that a lysine (K100) on the surface of CRP has a dual function: to promote CRP activity at Class II promoters, and to ensure proper CRP steady state levels. Both functions require the lysine's positive charge; intriguingly, the positive charge of K100 can be neutralized by acetylation using the central metabolite acetyl phosphate as the acetyl donor. We propose that CRP K100 acetylation could be a mechanism by which the cell downwardly tunes CRP-dependent Class II promoter activity, whilst elevating CRP steady state levels, thus indirectly increasing Class I promoter activity. This mechanism would operate under conditions that favor acetate fermentation, such as during growth on glucose as the sole carbon source or when carbon flux exceeds the capacity of the central metabolic pathways., (© 2017 John Wiley & Sons Ltd.)

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies. IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States., (Copyright © 2017 Post et al.)

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.

Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH ® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Background: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies., Methods and Results: Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming., Conclusions: These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Post-translational modification of lysine residues by N Ɛ -acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy. Graphical Abstract ᅟ.

Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human β-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism., (© 2015 John Wiley & Sons Ltd.)

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6-10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD), are neurodegenerative diseases in which tau fibrils accumulate. Recent evidence supports soluble tau species as the major toxic species. How soluble tau accumulates and causes neurodegeneration remains unclear. Here we identify tau acetylation at Lys174 (K174) as an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice. The acetyl-mimicking mutant K174Q slows tau turnover and induces cognitive deficits in vivo. Acetyltransferase p300-induced tau acetylation is inhibited by salsalate and salicylate, which enhance tau turnover and reduce tau levels. In the PS19 transgenic mouse model of FTD, administration of salsalate after disease onset inhibited p300 activity, lowered levels of total tau and tau acetylated at K174, rescued tau-induced memory deficits and prevented hippocampal atrophy. The tau-lowering and protective effects of salsalate were diminished in neurons expressing K174Q tau. Targeting tau acetylation could be a new therapeutic strategy against human tauopathies.

Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202-214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken β-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148)., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD(+)-dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5(-/-) mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5(-/-) animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5(-/-) compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mice are used extensively in transplantation studies involving bone marrow ablation. Due to the increasing security issues and expenses involved with γ irradiators, self-contained X-ray irradiators have been increasing in popularity. We hypothesized that bone marrow ablation by irradiation of mice with a (137)Cs irradiator would be comparable to that from an X-ray source irradiator. A lethal-dose curve was obtained by irradiating C57BL/6J mice with 500, 700, 900, and 1100 cGy from either source. These data were used to determine the lethal radiation exposure range for a noncompetitive bone marrow engraftment curve for each source. At 90 d after reconstitution, the bone marrow engraftment curves revealed significant differences between the 2 sources in the establishment of B cell, myeloid, and T cell lineages. Murine B cell reconstitution after exposure to a (137)Cs source was greater than that after X-ray exposure at each dose level, whereas the converse was true for myeloid cell reconstitution. At the 1050- and 1100-cGy doses, mice irradiated by using the X-ray source demonstrated higher levels of T cell reconstitution but decreased survival compared with mice irradiated with the (137)Cs source. We concluded that although both sources ablated endogenous bone marrow sufficiently to enable stem cell engraftment, there are distinct physiologic responses that should be considered when choosing the optimal source for use in a study and that irradiation from the (137)Cs source was associated with lower overall morbidity due to opportunistic infection.

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.

We report the implementation of high-quality signal processing algorithms into ProteoWizard, an efficient, open-source software package designed for analyzing proteomics tandem mass spectrometry data. Specifically, a new wavelet-based peak-picker (CantWaiT) and a precursor charge determination algorithm (Turbocharger) have been implemented. These additions into ProteoWizard provide universal tools that are independent of vendor platform for tandem mass spectrometry analyses and have particular utility for intralaboratory studies requiring the advantages of different platforms convergent on a particular workflow or for interlaboratory investigations spanning multiple platforms. We compared results from these tools to those obtained using vendor and commercial software, finding that in all cases our algorithms resulted in a comparable number of identified peptides for simple and complex samples measured on Waters, Agilent, and AB SCIEX quadrupole time-of-flight and Thermo Q-Exactive mass spectrometers. The mass accuracy of matched precursor ions also compared favorably with vendor and commercial tools. Additionally, typical analysis runtimes (∼1-100 ms per MS/MS spectrum) were short enough to enable the practical use of these high-quality signal processing tools for large clinical and research data sets.

N(ε) -lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε-amino group of a lysine residue can be acetylated either catalytically by acetyl-coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA-dependent N(ε) -lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD(+) -dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild-type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three-dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Purpose: The objective of the current study was to compare surgeon-rated visualization in shoulder arthroscopy using irrigation fluid with and without epinephrine., Methods: Eighty-three patients were randomized to receive irrigation fluid with (44 patients) or without (39 patients) epinephrine during their arthroscopic shoulder procedures. After each procedure, the blinded senior author (G.F.C.) evaluated visualization based on a visual analog scale (VAS), and all clinically important procedure variables were recorded., Results: Eighty-three arthroscopic shoulder procedures were included in the study. Fifty-four of these procedures were arthroscopic rotator cuff repairs, allowing a subset analysis of this specific procedure. There was a significant difference, with improved visualization in the epinephrine group versus the group without epinephrine when comparing all procedures (P < .0001) and when comparing only rotator cuff repairs (P < .0001). However, there was no statistical difference in other clinically important variables, including operative time and amount of irrigation fluid used., Conclusions: The addition of epinephrine to irrigation fluid significantly improves surgeon-rated visualization in shoulder arthroscopy. Without an observed significant difference in operative time or volume of irrigation fluid used, the clinical significance of this improved visualization is unclear, and the use of irrigation fluid without epinephrine remains a viable option in the hands of an experienced surgeon., Level of Evidence: Level I, high-quality randomized controlled trial with statistically significant difference., (Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Anticipatory grief is the process associated with grieving the loss of loved ones in advance of their inevitable death. Because anticipatory grief has been associated with a variety of outcomes, risk factors for this condition deserve closer consideration. Fifty-seven family members of terminally ill, hospice-eligible veterans receiving palliative care services completed measures assessing psychosocial factors and conditions. Elevated anticipatory grief was found in families characterized by relational dependency, lower education, and poor grief-specific support, who also experienced discomfort with closeness and intimacy, neuroticism, spiritual crisis, and an inability to make sense of the loss. Thus, in this sample, anticipatory grief appears to be part of a cluster of factors and associated distress that call for early monitoring and possible intervention.

Background: Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either "light" (L) isoleucine or "heavy" (H) (13)C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence., Results: Comparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A., Conclusions: The present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.

Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH-MS2 data-independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH-MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CVinter < 30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CVinter > 30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell-extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many late-onset proteotoxic diseases are accompanied by a disruption in homeostasis of metals (metallostasis) including iron, copper and zinc. Although aging is the most prominent risk factor for these disorders, the impact of aging on metallostasis and its role in proteotoxic disease remain poorly understood. Moreover, it is not clear whether a loss of metallostasis influences normal aging. We have investigated the role of metallostasis in longevity ofCaenorhabditis elegans. We found that calcium, copper, iron, and manganese levels increase as a function of age, while potassium and phosphorus levels tend to decrease. Increased dietary iron significantly accelerated the age-related accumulation of insoluble protein, a molecular pathology of aging. Proteomic analysis revealed widespread effects of dietary iron in multiple organelles and tissues. Pharmacological interventions to block accumulation of specific metals attenuated many models of proteotoxicity and extended normal lifespan. Collectively, these results suggest that a loss of metallostasis with aging contributes to age-related protein aggregation.