Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Simple urinary sample preparation for proteomic analysis.

Author(s): Khan A, Packer NH

Publication: J Proteome Res, 2006, Vol. 5, Page 2824-38

PubMed ID: 17022654 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the number of protein spots obtained though different precipitation methods and from urine collected at different times of day and during different weeks and also determined if the precipitation method used was compatible with protein identification through MALDI-TOF MS. Urine was collected from one healthy male over the course of 6 weeks at 5 AM (each week), 3 PM (3rd and 6th weeks) and at 10 PM (3rd week only), a protease inhibitor cocktail was added, and urine was frozen in 20 mL aliquots. Thawed aliquots were centrifuged at 1500 x g for 10 min at room temperature and proteins were precipitated by overnight incubation of 1 part urine to 5 parts solvent with one of the following solvents: acetone, ethanol, methanol, acetonitrile, or acetone and ethanol. Proteins were obtained from additional aliquots by ultrafiltration followed by acetone precipitation. After precipitation, proteins were sedimented by centrifugation at 4000 x g for 40 min at 10˚C. To investigate the effect of double precipitation, the Tris base was added to the supernatant from the acetonitrile precipitation to achieve a pH of 9.4 and the specimen was stored at -20˚C for 2.5 h before recentrifugation. Protein pellets were resuspended and reduced with 5 mM TBP and 10 mM acrylamide and desalted using a centrifugal filter. Finally, urine was rehydrated into immobiline pH gradient (IPG) strips or stored at -80 °C until analysis by 2-D gel electrophoresis followed by MALDI-TOF MS protein identification. The protocol was verified using a urine specimen from a patient with ovarian cancer.

   Summary of Findings:

   The greatest number of protein spots were found when specimens were precipitated with acetonitrile (567 spots) followed by ethanol (442 spots), methanol (365 spots), acetone and ethanol (324 spots), and acetone (233 spots). Further, ultrafiltration followed by acetone precipitation resulted in only 324 spots being detected. Although 707 spots were found in the first pellet and 133 in the second pellet in the double precipitation protocol using urine from a healthy subject, the 133 spots did not include any additional proteins. In contrast, when urine from a patient with ovarian cancer was used, 1098 spots were identified in the first precipitate and 722 in the second precipitate including some not identified during the first precipitation.  As expected, different spots were identified in the urine of the patient with ovarian cancer than in the healthy male patient.

   The intra-day variability was less than the inter-week variability. However, the spots identified changed slightly between collection timepoints within a day with significant differences in the number of proteins detected at 5 AM, 3 PM, and 10 PM (798, 703, and 729; respectfully, P=0.01). In comparison, 567, 672, 798 and 640 spots were detected in urine collected at 5 AM in the morning during the first, second, third, and fifth week, respectfully (P=0.01).

   A total of 395 proteins from 175 genes were identified by MALD-TOF-MS. Of the identified proteins, 19% were membrane-associated, 56% cytoplasmic or secreted, and 25% did not have a known subcellular localization. Further, 12% were kidney-specific, 25% were plasma specific, 12% were in both plasma and kidney, 9% were found in multiple tissues, 6% were in plasma and non-kidney tissues, 4% were ubiquitous, 24% were from non-kidney tissues, and 9% had unknown localization.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Ovarian cancer
   Analyte Extraction and Purification	Analyte isolation method	Acetone precipitation Ethanol precipitation Methanol precipitation Acetonitrile precipitation Acetone and ethanol precipitation Double acetonitrile precipitation at different pH Ultrafiltration and acetone precipitation
   Biospecimen Acquisition	Time of biospecimen collection	5 AM 3 PM 10 PM 1’st week 2'nd week 3'rd week 5'th week

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