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

Beretov J, Wasinger VC, Schwartz P, Graham PH, Li Y - Biomark Cancer (2014)

Bottom Line: A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery.Ultimately, efforts should be made to standardize urine preparation protocols.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins.

View Article: PubMed Central - PubMed

Affiliation: Cancer Care Centre, St George Hospital, Gray St, Kogarah, NSW, Australia. ; St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, NSW, Australia. ; SEALS, Anatomical Pathology, St George Hospital, Gray St, Kogarah, NSW, Australia.

ABSTRACT
A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis.

No MeSH data available.


Related in: MedlinePlus

The comparison of urinary protein precipitation methods on SDS-PAGE. The effects of precipitation techniques and centrifugation on urinary proteins were examined on healthy control (A–B, n = 18) and metastatic BC samples (C–D, n = 15). The details of the technique employed, total protein concentrations, and number of proteins identified by LC-MS/MS are summarized in Table 2.
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Related In: Results  -  Collection


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f1-bic-6-2014-021: The comparison of urinary protein precipitation methods on SDS-PAGE. The effects of precipitation techniques and centrifugation on urinary proteins were examined on healthy control (A–B, n = 18) and metastatic BC samples (C–D, n = 15). The details of the technique employed, total protein concentrations, and number of proteins identified by LC-MS/MS are summarized in Table 2.

Mentions: Our results clearly indicate that the quality and the variability of the urinary protein recovery were greatly affected by the preparation protocols used. Initially, precipitation methods 1–4 were performed at LSC, and no MS data was detected in these protein sample extracts (Table 2: A1–2, C1–3). Additionally, the excessive drag in the gel results from the metastatic BC urine samples (Fig. 1: C1–3) demonstrated a difference between the metastatic BC and the control samples (Fig. 1: A2). Therefore, additional desalting steps to clarify the sample further were required. To observe the optimal method for urine sample preparation for metastatic BC and healthy control urine samples, seven different urine precipitation techniques were compared. This information was used to determine the method that could provide the highest resolution on SDS-PAGE and the greatest number of urinary proteins detected with LC-MS/MS.


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

Beretov J, Wasinger VC, Schwartz P, Graham PH, Li Y - Biomark Cancer (2014)

The comparison of urinary protein precipitation methods on SDS-PAGE. The effects of precipitation techniques and centrifugation on urinary proteins were examined on healthy control (A–B, n = 18) and metastatic BC samples (C–D, n = 15). The details of the technique employed, total protein concentrations, and number of proteins identified by LC-MS/MS are summarized in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4219630&req=5

f1-bic-6-2014-021: The comparison of urinary protein precipitation methods on SDS-PAGE. The effects of precipitation techniques and centrifugation on urinary proteins were examined on healthy control (A–B, n = 18) and metastatic BC samples (C–D, n = 15). The details of the technique employed, total protein concentrations, and number of proteins identified by LC-MS/MS are summarized in Table 2.
Mentions: Our results clearly indicate that the quality and the variability of the urinary protein recovery were greatly affected by the preparation protocols used. Initially, precipitation methods 1–4 were performed at LSC, and no MS data was detected in these protein sample extracts (Table 2: A1–2, C1–3). Additionally, the excessive drag in the gel results from the metastatic BC urine samples (Fig. 1: C1–3) demonstrated a difference between the metastatic BC and the control samples (Fig. 1: A2). Therefore, additional desalting steps to clarify the sample further were required. To observe the optimal method for urine sample preparation for metastatic BC and healthy control urine samples, seven different urine precipitation techniques were compared. This information was used to determine the method that could provide the highest resolution on SDS-PAGE and the greatest number of urinary proteins detected with LC-MS/MS.

Bottom Line: A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery.Ultimately, efforts should be made to standardize urine preparation protocols.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins.

View Article: PubMed Central - PubMed

Affiliation: Cancer Care Centre, St George Hospital, Gray St, Kogarah, NSW, Australia. ; St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, NSW, Australia. ; SEALS, Anatomical Pathology, St George Hospital, Gray St, Kogarah, NSW, Australia.

ABSTRACT
A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis.

No MeSH data available.


Related in: MedlinePlus