In-Gel <sup>18</sup>O Labeling for Improved Identification of Proteins from 2-DE Gel Spots in Comparative Proteomic Experiments

Broedel, Oliver; Krause, Eberhard; Stephanowitz, Heike; Schuemann, Michael; Eravci, Murat; Weist, Stephanie; Brunkau, Cindy; Wittke, Janosch; Eravci, Selda; Baumgartner, Andreas

doi:10.1021/pr8010765

Cited by 7 publications

(5 citation statements)

References 32 publications

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“…This is consistent with earlier reported data on the efficient and complete incorporation of 18 O onto the C-terminus of tryptic peptides with high purity H 2 18 O (>97%) and complete drying of gel pieces prior to digestion and high concentrations of trypsin are used to drive the reaction. 9,42 Tryptic peptides from both the CRC and the normal samples are mixed together (CRC samples labeled with 18 O) immediately prior to injection. Three of the CCAP that had been identified by the targeted MRM approach (Hemoglobin Beta, Protocadherin 24 and Myeloperoxidase) were chosen for this study and compared for CRC patient 4 and normal volunteer 3.…”

Section: Resultsmentioning

confidence: 99%

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Nice²

2010

J. Proteome Res.

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Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in both men and women. The fecal occult blood test is currently the first line method for CRC screening but has an unacceptably low sensitivity and specificity. Improved screening tests are therefore urgently required for early stage CRC screening. We have described a hypothesis-driven approach for a rapid biomarker discovery process whereby selected proteins previously implicated as colorectal cancer-associated proteins (CCAP), which can potentially be shed into the feces from a colorectal tumor, are targeted for excision from 1D-SDS-PAGE based on their predicted molecular weight followed by directed identification and relative quantification using multiple reaction monitoring (MRM). This approach can significantly reduce the time for clinical assay development with the added advantage that many proteins will have been validated by previous in vitro and/or in vivo studies. Sixty potential CCAPs were selected from the literature and appropriate MRM conditions were established for measurement of proteotypic peptides. Nineteen of these proteins were detected in the feces from a patient with colorectal cancer. Relative quantitation of these 19 CCAP across 5 CRC patients and 5 healthy volunteers were carried out, revealing hemoglobin, myeloperoxidase, S100A9, filamin A and l-plastin to be present only in the feces of CRC patients.

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Section: Resultsmentioning

confidence: 99%

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Nice²

2010

J. Proteome Res.

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“…Common denaturing agents that have been used in proteolytic 18 O labeling workflow include urea [16][17][18] and the acid-cleavable surfactant RapiGest TM [19]. When gel electrophoresis (1D-PAGE or 2D-PAGE) is included in the workflow, sodium dodecyl sulfate (SDS) or other surfactants that are commonly used for 2D-gel electrophoresis can be used to extract proteins, because these surfactants can easily be removed by washing the gel bands/spots with an aqueous solution prior to the ingel digestion of proteins [20,21].…”

Section: Recent Technological Advancements A) Protein Extractionmentioning

confidence: 99%

“…The compatibility of SDS-PAGE with 18 O labeling, allows 18 O labeling to be used for quantifying lipid-associated proteins, like membrane proteins, which are efficiently solubilized by SDS. Protein fractionation by 2D-PAGE is also reported [20]. Applying the proteolytic 18 O labeling technique to the proteins fractionated by 2D-PAGE overcomes the intrinsic problem of 2D-PAGE that is the presence of multiple proteins in any spots.…”

Section: B) Protein Fractionationmentioning

confidence: 99%

Recent Technological Developments in Proteolytic 18O Labeling

Hajkova

Rao

Miyagi³

2011

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The proteolytic 18 O labeling method determines the relative ratios of individual proteins between two samples. This technique utilizes a protease and water (H 2 16 O and H 2 18 O) to produce labeled peptides; peptides in one sample incorporate 16 O by labeling in H 2 16 O, and the other sample incorporates 18 O by labeling in H 2 18O. Both samples are mixed in a 1:1 ratio and subjected to mass spectrometric analysis to identify and quantify the proteins from which the peptides originated. Technical issues in sample preparation and data processing prevented this method from becoming widely accepted in the field of quantitative proteomics, however these problems have been resolved and the technique is now rapidly gaining popularity. This review focuses on the recent technological developments that have improved the reliability and practicality of proteolytic 18 O labeling, including improved peptide labeling techniques, techniques to prevent proteasecatalyzed 18 O to 16 O back exchange reaction, mass spectrometry platforms suitable for this technique, computational tools for the calculation of 16 O/ 18 O-peptide ratios, and a new strategy that allows to compare a large number of samples.

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“…It is often utilized to investigate up-regulated proteins and down-regulated proteins in the comparative proteome. 20 In the present paper, the 18 O labeling technique was applied for the preparation of internal standards. To improve the selectivity and specificity, multiple reaction monitoring (MRM) mode in mass spectrometry was used to quantify the unique peptides of Cry1Ab and the corresponding 18 O-labeled peptides in the complex mixture.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution

Zhang

Lai

et al. 2012

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Cry1Ab is one of the most common Bacillus thuringiensis (Bt) proteins in genetically modified crops, which exhibits strong resistance against insect pests. In the present study, a sensitive and precise liquid chromatography stable isotope dilution multiple reaction monitoring tandem mass spectrometry (LC-SID-MRM-MS) assay was developed and validated to quantify the amount of Cry1Ab expression in transgenic maize leaves. The measurement of protein was converted to measurement of unique peptides to Cry1Ab protein. Two peptides unique to Cry1Ab were synthesized and labeled in H(2)(18)O to generate (18)O stable isotope peptides as internal standards. The validated method obtained superior specificity and good linearity. And the inter- and intra-day precision and accuracy for all samples were satisfactory. The results demonstrated Cry1Ab protein was 31.7 ± 4.1 μg g(-1) dry weight in Bt-176 transgenic maize leaves. It proved that the novel LC-SID-MRM-MS method was sensitive and selective to quantify Cry1Ab in the crude extract without time-consuming pre-separation or purification procedures.

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In-Gel ¹⁸O Labeling for Improved Identification of Proteins from 2-DE Gel Spots in Comparative Proteomic Experiments

Cited by 7 publications

References 32 publications

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Recent Technological Developments in Proteolytic 18O Labeling

Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution

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In-Gel 18O Labeling for Improved Identification of Proteins from 2-DE Gel Spots in Comparative Proteomic Experiments

Cited by 7 publications

References 32 publications

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Targeted In-Gel MRM: A Hypothesis Driven Approach for Colorectal Cancer Biomarker Discovery in Human Feces

Recent Technological Developments in Proteolytic 18O Labeling

Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution

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Product

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In-Gel ¹⁸O Labeling for Improved Identification of Proteins from 2-DE Gel Spots in Comparative Proteomic Experiments