Systematic evaluation of sample preparation methods for gel-based human urinary proteomics: quantity, quality, and variability.

Author(s): Thongboonkerd V, Chutipongtanate S, Kanlaya R

Publication: J Proteome Res, 2006, Vol. 5, Page 183

PubMed ID: 16396510 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to conduct a comparative assessment of 38 protocols for urinary protein isolation using 2D gel analysis.

   Summary of Findings:

   The resolution of 2D-gel electrophoretic spots was greater after sample dialysis. Of the 38 protocols evaluated for urinary protein isolation, precipitation with 90% ethanol resulted in the greatest protein yield (93%) and 10% acetic acid the lowest yield (2%). The authors recommend precipitation with ethanol, methanol, or acetone due to high protein yields, and the large number of protein spots observed with 2D gel electrophoresis. Spots themselves were protocol specific, with employment of more than one protocol resulting in an additive effect in terms of spot number.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Acetic acid at 10%, 20%, 50%, 75%, or 90% Acetone at 10%, 20%, 50%, 75%, or 90% Acetonitrile at 10%, 20%, 50%, 75%, or 90% Ammonium sulfate at 10%, 20%, 50%, 75%, or 90% Chloroform at 10%, 20%, 50%, 75%, or 90% Ethanol at 10%, 20%, 50%, 75%, or 90% Methanol at 10%, 20%, 50%, 75%, or 90% Centrifugal filtration Lyophilization Ultracentrifugation
   Analyte Extraction and Purification	Analyte isolation method	Dialysis conducted Dialysis not conducted

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2. Study Purpose

   The purpose of this study was to determine the influence of preacquisition (gender, caffeine or water intake) and acquisition (time of day of collection) variables on the recovery and profile of the urinary proteome.

   Summary of Findings:

   Water loading, rather than caffeine intake, had the greatest effect on the urinary proteome in terms of the number of unique spots observed with 2D gel analysis. Although evaluated in a single individual, urinary protein yield and the number of spots observed with 2D gel electrophoresis were influenced by the time of day of collection and fluid intake. The first morning urine sample was the most concentrated in terms of total protein quantity, although it yielded the fewest number of spots. Water loading yielded the lowest protein concentration and the second greatest number of spots.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	First morning urine Afternoon urine collection Urine collection after 1 cup of coffee Urine collection 20 min after 1L of water
   Preaquisition	Patient gender	Female Male

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