Quantitative Mass Spectrometry Identifies Insulin Signaling Targets in
            <i>C. elegans</i>

Dong, Meng‐Qiu; Venable, John D.; Au, Nora; Xu, Tao; Park, Sung Kyu; Cociorva, Daniel; Johnson, Jeffrey R.; Dillin, Andrew; Yates, John R.

doi:10.1126/science.1139952

Cited by 308 publications

(341 citation statements)

References 21 publications

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“…Proteins whose ratios differed from the normalized median by more than 2-fold and whose relative standard errors across replicates were less than 50% were considered differentially expressed (Table 2) (see ref. 21). We identified 10 differentially expressed proteins that met these criteria; other differentially expressed proteins with greater biological variability were seen also (Table S4).…”

Section: Proteomic Comparison Of Colonic Tumors and Normal Epitheliummentioning

confidence: 99%

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Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

Huttlin

Chen²,

Barrett-Wilt³

et al. 2009

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

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The unique biology of a neoplasm is reflected by its distinct molecular profile compared with normal tissue. To understand tumor development better, we have undertaken a quantitative proteomic search for abnormally expressed proteins in colonic tumors from Apc Min/؉ (Min) mice. By raising pairs of Min and wild-type mice on diets derived from natural-abundance or 15 Nlabeled algae, we used metabolic labeling to compare protein levels in colonic tumor versus normal tissue. Because metabolic labeling allows internal control throughout sample preparation and analysis, technical error is minimized as compared with in vitro labeling. Several proteins displayed altered expression, and a subset was validated via stable isotopic dilution using synthetic peptide standards. We also compared gene and protein expression among tumor and nontumor tissue, revealing limited correlation. This divergence was especially pronounced for species showing biological change, highlighting the complementary perspectives provided by transcriptomics and proteomics. Our work demonstrates the power of metabolic labeling combined with stable isotopic dilution as an integrated strategy for the identification and validation of differentially expressed proteins using rodent models of human disease.colon cancer ͉ min mouse ͉ proteomics ͉ differential mass spectrometry ͉ transcriptomics

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Section: Proteomic Comparison Of Colonic Tumors and Normal Epitheliummentioning

confidence: 99%

“…2 Each protein was quantified in multiple mouse pairs, with a mean ratio exceeding a 2-fold change and a relative standard error of less than 50% (see ref. 21). Mean microarray expression ratios also are listed for each protein.…”

Section: Proteomic Comparison Of Colonic Tumors and Normal Epitheliummentioning

confidence: 99%

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

Huttlin

Chen²,

Barrett-Wilt³

et al. 2009

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

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“…M uch progress has been made in mass spectrometry (MS)-based quantitative proteomics in recent years, as evidenced by numerous applications, such as biomarker discovery, 1 study of chromatin assembly and disassembly, 2 identification of insulin signaling targets, 3 and protein posttranslational modification (PTM). 4 Among the most commonly used quantitative strategies are full MS scan-based quantitation methods, such as SILAC (stable isotope labeling with amino acids in cell), 5 15 N-labeling, 6 and 18 O-labeling.…”

mentioning

confidence: 99%

pQuant Improves Quantitation by Keeping out Interfering Signals and Evaluating the Accuracy of Calculated Ratios

et al. 2014

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In relative protein abundance determination from peptide intensities recorded in full mass scans, a major complication that affects quantitation accuracy is signal interference from coeluting ions of similar m/z values. Here, we present pQuant, a quantitation software tool that solves this problem. pQuant detects interference signals, identifies for each peptide a pair of least interfered isotopic chromatograms: one for the light and one for the heavy isotope-labeled peptide. On the basis of these isotopic pairs, pQuant calculates the relative heavy/light peptide ratios along with their 99.75% confidence intervals (CIs). From the peptides ratios and their CIs, pQuant estimates the protein ratios and associated CIs by kernel density estimation. We tested pQuant, Census and MaxQuant on data sets obtained from mixtures (at varying mixing ratios from 10:1 to 1:10) of light-and heavy-SILAC labeled HeLa cells or 14 N-and 15 N-labeled Escherichia coli cells. pQuant quantitated more peptides with better accuracy than Census and MaxQuant in all 14 data sets. On the SILAC data sets, the nonquantified "NaN" (not a number) ratios generated by Census, MaxQuant, and pQuant accounted for 2.5−10.7%, 1.8−2.7%, and 0.01−0.5% of all ratios, respectively. On the 14 N/ 15 N data sets, which cannot be quantified by MaxQuant, Census and pQuant produced 0.9−10.0% and 0.3−2.9% NaN ratios, respectively. Excluding these NaN results, the standard deviations of the numerical ratios calculated by Census or MaxQuant are 30−100% larger than those by pQuant. These results show that pQuant outperforms Census and MaxQuant in SILAC and 15 N-based quantitation.M uch progress has been made in mass spectrometry (MS)-based quantitative proteomics in recent years, as evidenced by numerous applications, such as biomarker discovery, 1 study of chromatin assembly and disassembly, 2 identification of insulin signaling targets, 3 and protein posttranslational modification (PTM). 4 Among the most commonly used quantitative strategies are full MS scan-based quantitation methods, such as SILAC (stable isotope labeling with amino acids in cell), 5 15 N-labeling, 6 and 18 O-labeling. 7 In these strategies, proteins are metabolically labeled with stable isotopes, digested into peptides, and then analyzed using liquid chromatography (LC)-MS/MS. Quantitation software tools are designed to extract the intensities of pairs of light (L, unlabeled) and heavy (H, labeled) peptides from full MS scans. The relative abundance ratio of a protein between two conditions is then calculated based on the ratios of its constituent peptides. 8 For high-complexity samples such as whole cell lysates, it is not uncommon that a peptide coelutes with another peptide or a nonpeptide contaminant of a similar m/z value. 9 The interference caused by coeluting ions of similar m/z values can seriously compromise the accuracy of quantitation. 10,11 We examined two leading quantitation software tools Census 12 and MaxQuant, 13 and found that a lot of the peptide quantitation results ...

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“…Dissection of the insulin/IGF signalling pathway has identified sod-3 as a significant target for the DAF-16/FOXO transcription factor. Inhibition of the insulin/IGF signalling pathway results in elevated expression of sod-3 and an extension in lifespan (Murphy et al, 2003;Lee et al, 2003;Dong et al, 2007). Recently, it has been suggested that these MnSODs function in the insulin/IGF signalling pathway as physiological redox modulators rather than antioxidants (Honda et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Purification, crystallization and X-ray structures of the two manganese superoxide dismutases fromCaenorhabditis elegans

et al. 2008

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PDB References: manganese superoxide dismutase 3dc6, r3dc6sf; 3dc5, r3dc5sf. Caenorhabditis elegans expresses two manganese superoxide dismutase enzymes (MnSOD-2 and MnSOD-3) that are targeted to the mitochondrion. MnSOD-2 is constitutively expressed, while synthesis of MnSOD-3 is inducible. The structures of these two mononuclear metalloenzymes have been determined to 1.8 and 1.7 Å resolution, respectively. Pink crystals formed in space group P4 1 2 1 2 for each, with unit-cell parameters a = b = 81.0, c = 137.4 Å for MnSOD-2 and a = b = 81.8, c = 136.0 Å for MnSOD-3. The final structure of MnSOD-3 was refined to R = 21.6% and R free = 26.2% at 293 K, and R = 18.9% and R free = 22.6% at 100 K, while that of MnSOD-2 was refined to R = 16.9% and R free = 20.1% at 100 K. The asymmetric unit cell is comprised of two subunits. The resulting structures are very similar to that of human MnSOD and form a tetramer corresponding to a dimer of dimers. The subunit interface between dimers is comprised of two four-helix bundles that stabilize the biologically significant homotetramer.

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Quantitative Mass Spectrometry Identifies Insulin Signaling Targets in C. elegans

Cited by 308 publications

References 21 publications

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

pQuant Improves Quantitation by Keeping out Interfering Signals and Evaluating the Accuracy of Calculated Ratios

Purification, crystallization and X-ray structures of the two manganese superoxide dismutases fromCaenorhabditis elegans

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