Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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A standardized and reproducible urine preparation protocol for cancer biomarkers discovery.

Author(s): Beretov J, Wasinger VC, Schwartz P, Graham PH, Li Y

Publication: Biomark Cancer, 2014, Vol. 6, Page 21-7

PubMed ID: 25452700 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of protein purification protocol on the protein yield and identification of proteins by LC-MS/MS using urine specimens from 15 patients with metastatic breast cancer and 18 healthy patients.  Specimens were transported on ice and within 30 min were centrifuged at 2,000 x g at 4˚C for 10 min.  Supernatants were stored at -20˚C in 2 mL aliquots and transferred to -80˚C for long-term storage. Specimens were thawed fully and precipitated with acetone (1:8 for 1 h at -20˚C), TCA (4:1 for 1 h at 4˚C), or acetone (1:8 for 1 h at -20˚C) and then TCA (4:1 for 1 h at 4˚C), or were obtained by ultrafiltration using Amicon Ultra-15 Centrifu­gal Filter Units. After precipitation, pellets were either incubated with cetyl pyridinium chloride (CPC), cell-sheared (in lysis buffer at 5,000 rpm in a mini-bead beater), or left untreated. Following treatment specimens were then centrifuged at 11,000 x g for 30 min or were centrifuged at 4000 x g. Specimens were analyzed by SDS-PAGE and LC-MS/MS.

   Summary of Findings:

   The highest protein yields and the greatest number of proteins identified from both healthy patients and patients with metastatic breast cancer occurred when specimens were precipitated with both acetone and TCA and were centrifuged at 11,000 x g. Acetone precipitation alone resulted in slightly higher protein yields than TCA alone but the combined precipitation resulted in identification of approximately 50% more proteins than either precipitation method alone. Ultrafiltration resulted in low yields and identification of fewer proteins. The addition of cell-shearing or incubation in CPC after precipitation resulted in lower yields and the identification of fewer proteins.  Centrifugation at 11,000 x g was required to desalt and remove insoluble materials and specimens centrifuged at 4000 x g after precipitation had no proteins identified by LC-MS/MS. SDS-gels showed more drag in specimens from patients with metastatic breast cancer than in specimens from healthy patients. 

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   Protein	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Acetone precipitated TCA precipitated Acetone and TCA precipitated Ultrafiltration
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared
   Analyte Extraction and Purification	Analyte purification	CPC precipitation No CPC precipitation Cell-shearing No cell-shearing

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