When less can yield more – Computational preprocessing of MS/MS spectra for peptide identification

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258

313

Protein S-acylation (palmitoylation), a reversible posttranslational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomics approach, designated palmitoyl protein identification and site characterization (PalmPISC), for proteome scale enrichment and characterization of Sacylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be Sacylated on three cysteine residues within a novel CCX 7-13 C(S/T) motif downstream of a conserved Asp-HisHis-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-Acylation may tether cytoplasmic acylprotein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatics analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large scale human Sacylated protein data set are expected to provide powerful tools to facilitate our understanding of the functions and mechanisms of protein S-acylation. Molecular & Cellular Proteomics 9:54 -70, 2010.Protein S-acylation, commonly but somewhat inaccurately known as protein palmitoylation, is a post-translational lipid modification involving the covalent addition of long-chain fatty acids (predominantly the 16-carbon palmitic acid) to protein cysteine thiols via thioester linkages (1). Like other lipid modifications such as myristoylation and prenylation, S-acylation increases the hydrophobicity of cytoplasmic proteins, including many signaling molecules, thereby increasing their affinity for cytosolic membrane surfaces. However, compared with myristoylation and prenylation, S-acylation is more frequently detected on transmembrane proteins such as G proteincoupled receptors, immune cell receptors, and ion channels, all of which are already tightly associated with membranes (2). Moreover, protein S-acylation, which may either occur spontaneously or be catalyzed by palmitoyl acyltransferases (PATs), 1 can be reversed by protein thioesterases such as acyl-protein thioest...

Section: Methodsmentioning

confidence: 98%

Proteome Scale Characterization of Human S-Acylated Proteins in Lipid Raft-enriched and Non-raft Membranes

Yang

Vizio

Kirchner

et al. 2010

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258

313

“…The ion trap was operated in datadependent acquisition mode, fragmenting up to six of the most intensive ions after each survey scan. The Thermo ".raw" files were converted into the Mascot generic format (mgf) by extracting the 200 most intense fragment ions for each MS/MS spectrum (14).…”

Section: Proteomic Analysis For Discovery Stage 1-gelc-ms/ms Methodmentioning

confidence: 99%

Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome

Broadbelt

Rivera

Paterson

et al. 2012

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Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS E -based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p < 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT 1A receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.009530, 1-17, 2012. The sudden infant death syndrome (SIDS)1 is the sudden unexpected death of an infant less than 1 year of age, with onset of the fatal episode apparently occurring during sleep, that remains unexplained after a thorough investigation, including performance of a complete autopsy and review of the circumstances of death and clinical history (1). It is the leading cause of postneonatal infant mortality in the United States today; with an overall incidence of 0.53/1000 live birth(s) (2). Typically, a seemingly healthy infant is found dead after a sleep period, either in the early morning or after a day-time nap (1,3,4). Impaired brainstem responses to homeostatic challenges during sleep may result in the sleep-related sudden death characteristic of SIDS (4). We have reported various abnormalities in serotonergic (5-HT) receptors, 5-HT transporter, and 5-HT cellular maturation in the medullary 5-HT system in SIDS cases in three independent data sets over the last decade (5-7). This system within the medulla oblongata is a neural network comprised of 5-HT source neurons and their projection sites that help mediate homeostatic responses during the sleep-wake cycle (3). The medullary 5-HT system is defined by us as the regions of t...

“…Database Searching-Peak lists of the 200 most abundant fragment ions from each product ion spectrum were extracted out of Thermo .raw files and converted into .mgf files using in-house software (21). No further data processing such as smoothing, de-isotoping, and/or filtering were carried out.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

Yang

Chung

Kim

et al. 2011

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Antiproliferative factor (APF), a Frizzled-8 protein-related sialoglycopeptide involved in the pathogenesis of interstitial cystitis, potently inhibits proliferation of normal urothelial cells as well as certain cancer cells. To elucidate the molecular mechanisms of the growth-inhibitory effect of APF, we performed stable isotope labeling by amino acids in cell culture analysis of T24 bladder cancer cells treated with and without APF. Among over 2000 proteins identified, 54 were significantly up-regulated and 48 were down-regulated by APF treatment. Bioinformatic analysis revealed that a protein network involved in cell adhesion was substantially altered by APF and that ␤-catenin was a prominent node in this network. Functional assays demonstrated that APF down-regulated ␤-catenin, at least in part, via proteasomal and lysosomal degradation. Moreover, silencing of ␤-catenin mimicked the antiproliferative effect of APF whereas ectopic expression of nondegradable ␤-catenin rescued growth inhibition in response to APF, confirming that ␤-catenin is a key mediator of APF signaling. Notably, the key role of ␤-catenin in APF signaling is not restricted to T24 cells, but was also observed in an hTERT-immortalized human bladder epithelial cell line, TRT-HU1. In addition, the network model suggested that ␤-catenin is linked to cyclooxygenase-2 (COX-2), implying a potential connection between APF and inflammation. Functional assays verified that APF increased the production of prostaglandin E 2 and that down-modulation of ␤-catenin elevated COX-2 expression, whereas forced expression of nondegradable ␤-catenin inhibited APF-induced up-regulation of COX-2. Furthermore, we confirmed that ␤-catenin was down-regulated whereas COX-2 was up-regulated in epithelial cells explanted from IC bladder biopsies compared with control tissues. In summary, our quantitative proteomics study describes the first provisional APF-regulated protein network, within which ␤-catenin is a key node, and provides new insight that targeting the ␤-catenin signaling pathway may be a rational approach toward treating interstitial cystitis. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.007492, 1-11, 2011. Antiproliferative factor (APF)1 , a nine-residue sialoglycopeptide whose peptide chain is 100% homologous to the putative sixth transmembrane domain of , is secreted by bladder epithelial cells from patients with interstitial cystitis (IC) (2, 3), a prevalent and debilitating pelvic disorder (4, 5). Studies suggest that APF is a potent negative growth factor, which markedly inhibits the proliferation of not only normal bladder epithelial cells but also T24 bladder carcinoma cells and HeLa cervical carcinoma cells (1, 6, 7). Studies have been conducted to investigate the molecular mechanisms underlying the antiproliferative effect of APF using the hypothesis-driven approach; these led to the discoveries that (a) cytoskeleton-associated protein 4 (CKAP4), also known as CLIMP63, is a high-affinity receptor for APF (6); (b) palmitoylation of CKAP4 by ...