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Differential proteome profiling using iTRAQ in microalbuminuric and normoalbuminuric type 2 diabetic patients.

Jin J, Ku YH, Kim Y, Kim Y, Kim K, Lee JY, Cho YM, Lee HK, Park KS, Kim Y - Exp Diabetes Res (2012)

Bottom Line: Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM).Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921.Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, 28 Yongon-Dong, Seoul 110-799, Republic of Korea.

ABSTRACT
Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values (0.849, 0.873, and 0.825, resp.). Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.

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Correlation between the 2 technical replicates and determination of the cutoff value for significant fold changes. (a) Plots of iTRAQ ratios for two technical replicates. Forty-four proteins were commonly observed from technical replicate 1 (labeled 115/114), and 26 proteins were commonly observed from technical replicate 2 (labeled 117/116). These differentially excreted proteins (P value < 0.05, more than two unique peptides: >95%) were plotted in the linear dynamic range. The technical variations yielded a correlation coefficient of r2 = 0.9527 and r2 = 0.9178 between iTRAQ experiments 1 and 2, respectively. (b) The % variations for the common proteins from the two technical replicates. The 44 and 26 common proteins from the 2 technical replicates were used as inputs to calculate % variations. The vertical axis represents the number of proteins, and the horizontal axis denotes % variation. Ninety percent of the proteins fell within 25% of the respective experimental variation. Thus, we considered a fold-change of >1.25 or <0.80, a meaningful cutoff that represented actual differences in the iTRAQ experiments.
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fig2: Correlation between the 2 technical replicates and determination of the cutoff value for significant fold changes. (a) Plots of iTRAQ ratios for two technical replicates. Forty-four proteins were commonly observed from technical replicate 1 (labeled 115/114), and 26 proteins were commonly observed from technical replicate 2 (labeled 117/116). These differentially excreted proteins (P value < 0.05, more than two unique peptides: >95%) were plotted in the linear dynamic range. The technical variations yielded a correlation coefficient of r2 = 0.9527 and r2 = 0.9178 between iTRAQ experiments 1 and 2, respectively. (b) The % variations for the common proteins from the two technical replicates. The 44 and 26 common proteins from the 2 technical replicates were used as inputs to calculate % variations. The vertical axis represents the number of proteins, and the horizontal axis denotes % variation. Ninety percent of the proteins fell within 25% of the respective experimental variation. Thus, we considered a fold-change of >1.25 or <0.80, a meaningful cutoff that represented actual differences in the iTRAQ experiments.

Mentions: The technical variations for the 115/114 and 117/116 reporter ions, calculated using the ratios of the 44 and 26 commonly observed proteins between the 2 technical replicates, were r2 = 0.9527 and r2 = 0.9178, respectively (Figure 2(a)). Accordingly, 90% of the commonly observed in the technical replicates fell within 25% of the respective experimental variation (Figure 2(b)). Therefore, we set fold-change thresholds of >1.25 or <0.80 to identify true differences between the expression of 115/114 and 116/117 reporter ions, as described in [23].


Differential proteome profiling using iTRAQ in microalbuminuric and normoalbuminuric type 2 diabetic patients.

Jin J, Ku YH, Kim Y, Kim Y, Kim K, Lee JY, Cho YM, Lee HK, Park KS, Kim Y - Exp Diabetes Res (2012)

Correlation between the 2 technical replicates and determination of the cutoff value for significant fold changes. (a) Plots of iTRAQ ratios for two technical replicates. Forty-four proteins were commonly observed from technical replicate 1 (labeled 115/114), and 26 proteins were commonly observed from technical replicate 2 (labeled 117/116). These differentially excreted proteins (P value < 0.05, more than two unique peptides: >95%) were plotted in the linear dynamic range. The technical variations yielded a correlation coefficient of r2 = 0.9527 and r2 = 0.9178 between iTRAQ experiments 1 and 2, respectively. (b) The % variations for the common proteins from the two technical replicates. The 44 and 26 common proteins from the 2 technical replicates were used as inputs to calculate % variations. The vertical axis represents the number of proteins, and the horizontal axis denotes % variation. Ninety percent of the proteins fell within 25% of the respective experimental variation. Thus, we considered a fold-change of >1.25 or <0.80, a meaningful cutoff that represented actual differences in the iTRAQ experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3318901&req=5

fig2: Correlation between the 2 technical replicates and determination of the cutoff value for significant fold changes. (a) Plots of iTRAQ ratios for two technical replicates. Forty-four proteins were commonly observed from technical replicate 1 (labeled 115/114), and 26 proteins were commonly observed from technical replicate 2 (labeled 117/116). These differentially excreted proteins (P value < 0.05, more than two unique peptides: >95%) were plotted in the linear dynamic range. The technical variations yielded a correlation coefficient of r2 = 0.9527 and r2 = 0.9178 between iTRAQ experiments 1 and 2, respectively. (b) The % variations for the common proteins from the two technical replicates. The 44 and 26 common proteins from the 2 technical replicates were used as inputs to calculate % variations. The vertical axis represents the number of proteins, and the horizontal axis denotes % variation. Ninety percent of the proteins fell within 25% of the respective experimental variation. Thus, we considered a fold-change of >1.25 or <0.80, a meaningful cutoff that represented actual differences in the iTRAQ experiments.
Mentions: The technical variations for the 115/114 and 117/116 reporter ions, calculated using the ratios of the 44 and 26 commonly observed proteins between the 2 technical replicates, were r2 = 0.9527 and r2 = 0.9178, respectively (Figure 2(a)). Accordingly, 90% of the commonly observed in the technical replicates fell within 25% of the respective experimental variation (Figure 2(b)). Therefore, we set fold-change thresholds of >1.25 or <0.80 to identify true differences between the expression of 115/114 and 116/117 reporter ions, as described in [23].

Bottom Line: Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM).Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921.Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, 28 Yongon-Dong, Seoul 110-799, Republic of Korea.

ABSTRACT
Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values (0.849, 0.873, and 0.825, resp.). Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.

Show MeSH
Related in: MedlinePlus