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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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Related in: MedlinePlus

Workflow of iTRAQ, 2-DE, Western blot, and MRM of the urinary proteome. For analysis of the urinary proteome, 3 iTRAQ experiments were performed, 2-DE, Western blot, and MRM were conducted to confirm and validate the iTRAQ results. iTRAQ experiments 1, 2, and 3 were performed, labeled (a) and (b), (c) and (d), and (e), respectively, wherein 3 biological replicates (labeled (a), (d), and (e), resp.), technical replicate 1 (labeled (a) and (c)), and technical replicate 2 (labeled (b) and (d)) were performed in microalbuminuric versus normoalbuminuric urine. 2-DE, Western blot, and MRM analysis of the urinary proteome were conducted using labeled (f), (g), and (h), respectively.
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fig1: Workflow of iTRAQ, 2-DE, Western blot, and MRM of the urinary proteome. For analysis of the urinary proteome, 3 iTRAQ experiments were performed, 2-DE, Western blot, and MRM were conducted to confirm and validate the iTRAQ results. iTRAQ experiments 1, 2, and 3 were performed, labeled (a) and (b), (c) and (d), and (e), respectively, wherein 3 biological replicates (labeled (a), (d), and (e), resp.), technical replicate 1 (labeled (a) and (c)), and technical replicate 2 (labeled (b) and (d)) were performed in microalbuminuric versus normoalbuminuric urine. 2-DE, Western blot, and MRM analysis of the urinary proteome were conducted using labeled (f), (g), and (h), respectively.

Mentions: In this study, 3 iTRAQ experiments were performed. The detailed iTRAQ labeling strategy is summarized for the specified NA/MA urine samples in Figure 1 and Table 1; iTRAQ Experiments 1, 2, and 3 were performed for labeling (a) and (b), (c) and (d), and (e), respectively. Each normoalbuminuric peptide was labeled with iTRAQ reagents 114, 115, and 116, and the microalbuminuric peptide was labeled with iTRAQ reagents 115 and 117 (Figure 1). The 2 sample sets (microalbuminuric and normoalbuminuric) were combined and dried. To analyze the proteome quantitatively using iTRAQ labeling, we determined the labeling efficiency, as described [23]; the number of possible labeling sites (the N-termini of all peptides and lysine side chains) in 21,610 peptides were compared manually with that of completely labeled sites, represented by the Pro GroupTM Algorithm in ProteinPilot.


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)

Workflow of iTRAQ, 2-DE, Western blot, and MRM of the urinary proteome. For analysis of the urinary proteome, 3 iTRAQ experiments were performed, 2-DE, Western blot, and MRM were conducted to confirm and validate the iTRAQ results. iTRAQ experiments 1, 2, and 3 were performed, labeled (a) and (b), (c) and (d), and (e), respectively, wherein 3 biological replicates (labeled (a), (d), and (e), resp.), technical replicate 1 (labeled (a) and (c)), and technical replicate 2 (labeled (b) and (d)) were performed in microalbuminuric versus normoalbuminuric urine. 2-DE, Western blot, and MRM analysis of the urinary proteome were conducted using labeled (f), (g), and (h), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3318901&req=5

fig1: Workflow of iTRAQ, 2-DE, Western blot, and MRM of the urinary proteome. For analysis of the urinary proteome, 3 iTRAQ experiments were performed, 2-DE, Western blot, and MRM were conducted to confirm and validate the iTRAQ results. iTRAQ experiments 1, 2, and 3 were performed, labeled (a) and (b), (c) and (d), and (e), respectively, wherein 3 biological replicates (labeled (a), (d), and (e), resp.), technical replicate 1 (labeled (a) and (c)), and technical replicate 2 (labeled (b) and (d)) were performed in microalbuminuric versus normoalbuminuric urine. 2-DE, Western blot, and MRM analysis of the urinary proteome were conducted using labeled (f), (g), and (h), respectively.
Mentions: In this study, 3 iTRAQ experiments were performed. The detailed iTRAQ labeling strategy is summarized for the specified NA/MA urine samples in Figure 1 and Table 1; iTRAQ Experiments 1, 2, and 3 were performed for labeling (a) and (b), (c) and (d), and (e), respectively. Each normoalbuminuric peptide was labeled with iTRAQ reagents 114, 115, and 116, and the microalbuminuric peptide was labeled with iTRAQ reagents 115 and 117 (Figure 1). The 2 sample sets (microalbuminuric and normoalbuminuric) were combined and dried. To analyze the proteome quantitatively using iTRAQ labeling, we determined the labeling efficiency, as described [23]; the number of possible labeling sites (the N-termini of all peptides and lysine side chains) in 21,610 peptides were compared manually with that of completely labeled sites, represented by the Pro GroupTM Algorithm in ProteinPilot.

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