2004
DOI: 10.1093/bioinformatics/bth446
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Quantifying reproducibility for differential proteomics: noise analysis for protein liquid chromatography-mass spectrometry of human serum

Abstract: Using replicated human serum samples, we applied an error model for proteomic differential expression profiling for a high-resolution liquid chromatography-mass spectrometry (LC-MS) platform. The detailed noise analysis presented here uses an experimental design that separates variance caused by sample preparation from variance due to analytical equipment. An analytic approach based on a two-component error model was applied, and in combination with an existing data driven technique that utilizes local sample … Show more

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Cited by 126 publications
(145 citation statements)
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“…The depleted CSF proteome was denatured, reduced, cysteines alkylated and after buffer exchange, digested, desalted, and fractionated into three fractions on a strong cation-exchange (SCX) column as previously described. [16][17][18][19][20] A total of approximately 20 μg of processed peptides dissolved in 40 μL 0.1% formic acid per SCX fraction were injected into the LC/MS system.…”
Section: Sample Preparationmentioning
confidence: 99%
See 1 more Smart Citation
“…The depleted CSF proteome was denatured, reduced, cysteines alkylated and after buffer exchange, digested, desalted, and fractionated into three fractions on a strong cation-exchange (SCX) column as previously described. [16][17][18][19][20] A total of approximately 20 μg of processed peptides dissolved in 40 μL 0.1% formic acid per SCX fraction were injected into the LC/MS system.…”
Section: Sample Preparationmentioning
confidence: 99%
“…[16][17][18][19][20] Molecular ion signal intensities were normalized on a global basis according to each samples' total protein content (after abundant protein depletion). For protein identification, approximately 500 proteins were identified by tandem MS (Model LTQ, Thermo Electron, San Jose, CA) using control CSF samples with an identification criteria score of more than 40 by Mascot software (Matrix Science, London, UK).…”
Section: Lc/ms Differential Quantification and Identificationmentioning
confidence: 99%
“…However, in practice, the reliability of the peak ratio depends on many experimental parameters. For protein quantitation in the proteomic scale, the variation in experimental results can be caused by the sample preparation process and the performance of the equipment used for analysis 34 . Although quantitation errors may be caused by the poor S/N ratios of low abundance peptides and overlapping of isotopic profiles, it is inevitable that errors will arise because of the limited dynamic range of the instrument used.…”
Section: Step 32 Determination and Application Of A Dynamic Range Fimentioning
confidence: 99%
“…It should also be noted that the dependence of quantification accuracy on signal intensity is not specific to iTRAQ analysis. Isotope coded affinity tag (ICAT) [33], LC/MS [34] and microarray [35,36] quantification measurements all share similar features. Thus, in order to obtain more accurate peptide expression ratios, we decided to filter out measurements less than 5000 RPAs.…”
Section: Quantification Accuracy Is Itraq Ion Intensity-dependentmentioning
confidence: 99%