Spectral Index for Assessment of Differential Protein Expression in Shotgun Proteomics

Fu, Xiaoyun; Gharib, Sina A.; Green, Pattie S.; Aitken, Moira L.; Frazer, David; Park, David R.; Vaisar, Tomáš; Heinecke, Jay W.

doi:10.1021/pr070271+

Cited by 98 publications

(127 citation statements)

References 45 publications

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“…Database searching was carried out on the IPI_rat_decoy database (IPI_Rat v3.48). Spectral count data from lysosomeenriched or lysosome-depleted samples were merged for semiquantitative analysis of the fractions (65), and statistical analysis was carried out according to the method of Fu et al (35) for enrichment evaluation. Oocytes were prepared and injected with 50 ng of PQLC2-EGFP, PQLC2-LL/ AA-EGFP, or EGFP cRNA as described (18).…”

Section: Methodsmentioning

confidence: 99%

“…The statistical significance of the association of PQLC2 with lysosomes was assessed by calculating for each identified protein a spectral index ranging from −1 to +1 for proteins exclusively detected in lysosome-depleted and lysosome-enriched fractions, respectively. This index combines the relative peptide abundance in tandem MS (MS/MS) spectra (spectral counts) and the number of samples with detectable peptides to provide an estimate of protein abundance (35). Across three biological replicates, we detected three peptides matching the rat PQLC2 sequence.…”

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confidence: 99%

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Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Jézégou

Llinares

Anne

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

155

184

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Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1–3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Jézégou

Llinares

Anne

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

155

184

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“…A scatter plot of peptide counts revealed a linear correlation between young and senescent cells, as well as differentially expressed proteins ( Fig. 2A) (Fu et al, 2008). We aligned the peptide counts of each protein based on the difference in peptide number between senescent cells and young cells; the converted graph represents the relative protein abundance in young and senescent cells (Fig.…”

Section: Comparative Analysis Of Young and Senescent Hdf Cellsmentioning

confidence: 94%

Differential Expression of Extracellular Matrix Proteins in Senescent and Young Human Fibroblasts: a Comparative Proteomics and Microarray Study

Yang

Kwon

Rhim

et al. 2011

Molecules and Cells

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The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.

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“…Importantly, the CAD subjects were newly diagnosed (though at least 3 months past any acute CAD event) and not on medications that alter lipid levels (see below). Using statistical approaches based on spectral counting [the number of MS/MS spectra unique to a protein (25,26)] and random permutation analysis (20,27), we found five proteins that were apparently enriched in HDL isolated from the CAD subjects. These proteins related to lipid metabolism (apoE, apoC-IV, and apoA-IV), oxidative stress (paraxonase-1), and the immune system (complement factor C3).…”

Section: Hdl Of Humans With Established Cad Has a Distinct Proteomic mentioning

confidence: 99%

The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

Heinecke

2009

Journal of Lipid Research

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One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL 3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy.-Heinecke, J. W. The HDL proteome: a marker-and perhaps mediator-of coronary artery disease. J. Lipid Res. 2009. 50: S167-S171.Supplementary key words high density lipoprotein • apolopoprotein E • oxidized high density lipoprotein • C-reactive protein • apolopoprotein A-I • clusterin HDL and LDL are the major carriers of cholesterol in human blood (1-3). Epidemiological, genetic, and clinical studies demonstrate that elevated levels of LDL or low levels of HDL are important risk factors for coronary artery disease (CAD). In striking contrast, high levels of HDL are cardioprotective. LDL promotes heart disease by delivering cholesterol to macrophages, a key early event in atherogenesis (1-3). HDL protects against atherosclerosis by removing cholesterol from artery wall macrophages by a process termed reverse cholesterol transport.HDL exhibits other biological activities that may contribute to its anti-atherogenic properties, such as the ability to inhibit inflammation (4). It has been proposed that its cardioprotective effects depend on the types of particles generated in vivo and that HDL in humans with established CAD is dysfunctional (4). Indeed, animal studies convincingly demonstrate that changes in proteins involved in HDL metabolism can promote atherosclerosis, even when plasma levels of HDL-cholesterol are elevated (5, 6). Moreover, in vitro studies demonstrate that oxidative damage impairs the ability of apolipoprotein A-I (apoA-I), the major HDL protein, to remove cholesterol from macrophages (7).One possible con...

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Spectral Index for Assessment of Differential Protein Expression in Shotgun Proteomics

Cited by 98 publications

References 45 publications

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Differential Expression of Extracellular Matrix Proteins in Senescent and Young Human Fibroblasts: a Comparative Proteomics and Microarray Study

The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

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