Optimizing a proteomics platform for urine biomarker discovery.

Author(s): Afkarian M, Bhasin M, Dillon ST, Guerrero MC, Nelson RG, Knowler WC, Thadhani R, Libermann TA

Publication: Mol Cell Proteomics, 2010, Vol. 9, Page 2195-204

PubMed ID: 20511394 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of protein concentration, protease inhibition, extraction method, and label amount on protein identification and quantification in urine specimens from 2 healthy individuals and 8 diabetic patients with or without nephropathy using iTRAQ. Urine from healthy individuals was frozen at -80 degrees C for up to 2 weeks prior to analysis. Urine from diabetic patients was stored at -80 degrees C for 9-17 years.

   Summary of Findings:

   Decreasing the amount of protein from 50 ug to 20 ug did not affect identification of proteins, but when 10 ug was used there was a 29% drop in proteins identified with high confidence and lower iTRAQ values. Decreasing the iTRAQ label to only 25% of normal and adding protease inhibitors did not affect protein identification or quantification. The highest protein yield and the most iTRAQ identified proteins were obtained from specimens extracted by methanol precipitation, but acetonitrile precipitation generated the most protein spots on 2D gels. More than 90% of proteins identified by iTRAQ were found in all specimens regardless of extraction method, but only methanol precipitate specimens contained all of the proteins identified by the other methods. The relative protein concentrations were dependent on extraction method so consistent extraction method was essential for specimen comparisons. The same 80 proteins were identified in depleted and non-depleted urine from a healthy individual, but 75 and 77 proteins were identified in depleted and non-depleted urine from a diabetic patient. Of the proteins identified in the urine from the diabetic patient, 73 were common between the two samples. Using the authors protocol, hierarchical clustering of 54 proteins identified by iTRAQ was able to distinguish specimens from patients with diabetes and those without nephropathy and proteins differentially expressed in the two conditions were identified.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Diabetes Type 2
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	MALDI-TOF MS
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Type II diabetes with nephropathy Type II diabetes without nephropathy Normal
   Analyte Extraction and Purification	Protease inhibitor	Cocktail No protease inhibitor added
   Biospecimen Aliquots and Components	Aliquot size/volume	10 ug protein 20 ug protein 50 ug protein
   Analyte Extraction and Purification	Analyte isolation method	Methanol precipitation Ethanol precipitation Acetonitrile precipitation Trichloroacetic acid precipitation Ultrafilteration Dialysis and lyophilization
   MALDI-TOF MS Specific	Detection method	Normal iTRAQ label 25% iTRAQ label
   Biospecimen Aliquots and Components	Biospecimen components	Not depleted Albumin/IgG depleted

   View more