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Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

Liu B, Qiu FH, Voss C, Xu Y, Zhao MZ, Wu YX, Nie J, Wang ZL - Proteome Sci (2011)

Bottom Line: Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots.During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels.Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, PR China. niejing@mail.sysu.edu.cn.

ABSTRACT

Background: High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum.

Results: The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins.

Conclusions: The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.

No MeSH data available.


Related in: MedlinePlus

Two Dimensional Electrophoresis Gel image of crude and depleted umbilical cord serum samples. For each gel, 150 μg of protein was separated over pH range 4-7 (24 cm strips) and 12.5% SDS-polyacrylamide gel. The gel was visualized by silver staining. The Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) Kit was used to deplete albumin and IgG in the umbilical cord serum. Removal of these proteins improved the resolution in the albumin and IgG areas on the gel and increased the intensity of low abundance proteins. A) Two Dimension Electrophoresis Gel image of crude umbilical cord serum. B) Two Dimension Electrophoresis Gel image of depleted umbilical cord serum by Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: regions of gel containing albumin, heavy and light chain of IgG before (panel A) and after depletion (panel B). Rectangle: used to highlight different protein patterns in the two groups, indicating a large number of protein spots that emerged in the depleted gel.
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Figure 2: Two Dimensional Electrophoresis Gel image of crude and depleted umbilical cord serum samples. For each gel, 150 μg of protein was separated over pH range 4-7 (24 cm strips) and 12.5% SDS-polyacrylamide gel. The gel was visualized by silver staining. The Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) Kit was used to deplete albumin and IgG in the umbilical cord serum. Removal of these proteins improved the resolution in the albumin and IgG areas on the gel and increased the intensity of low abundance proteins. A) Two Dimension Electrophoresis Gel image of crude umbilical cord serum. B) Two Dimension Electrophoresis Gel image of depleted umbilical cord serum by Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: regions of gel containing albumin, heavy and light chain of IgG before (panel A) and after depletion (panel B). Rectangle: used to highlight different protein patterns in the two groups, indicating a large number of protein spots that emerged in the depleted gel.

Mentions: As shown in Figure 2, albumin and IgG emerged in a large smeared protein pattern, masking proteins in a substantial area (Figure 2A, circles) on crude serum 2DE gels. Depletion of albumin and IgG by the PROTIA-Sigma kit improved the quality of 2DE gels in two ways: firstly, depletion of albumin and IgG resulted in improved separation within these regions of the gel and the emergence of several new spots (Figure 2B, circles). Additionally, the depletion of the high abundance proteins increased the load of low abundance proteins. Albumin and IgG represent about 80% of the total serum protein content and the PROTIA-Sigma kit was able to remove more than 90% of this albumin and IgG from the umbilical cord serum. Therefore, the loading capacity of low abundance proteins increased after protein depletion and many new protein spots emerged on the depleted serum 2DE gels (Figure 2B, Rectangle).


Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

Liu B, Qiu FH, Voss C, Xu Y, Zhao MZ, Wu YX, Nie J, Wang ZL - Proteome Sci (2011)

Two Dimensional Electrophoresis Gel image of crude and depleted umbilical cord serum samples. For each gel, 150 μg of protein was separated over pH range 4-7 (24 cm strips) and 12.5% SDS-polyacrylamide gel. The gel was visualized by silver staining. The Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) Kit was used to deplete albumin and IgG in the umbilical cord serum. Removal of these proteins improved the resolution in the albumin and IgG areas on the gel and increased the intensity of low abundance proteins. A) Two Dimension Electrophoresis Gel image of crude umbilical cord serum. B) Two Dimension Electrophoresis Gel image of depleted umbilical cord serum by Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: regions of gel containing albumin, heavy and light chain of IgG before (panel A) and after depletion (panel B). Rectangle: used to highlight different protein patterns in the two groups, indicating a large number of protein spots that emerged in the depleted gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105942&req=5

Figure 2: Two Dimensional Electrophoresis Gel image of crude and depleted umbilical cord serum samples. For each gel, 150 μg of protein was separated over pH range 4-7 (24 cm strips) and 12.5% SDS-polyacrylamide gel. The gel was visualized by silver staining. The Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) Kit was used to deplete albumin and IgG in the umbilical cord serum. Removal of these proteins improved the resolution in the albumin and IgG areas on the gel and increased the intensity of low abundance proteins. A) Two Dimension Electrophoresis Gel image of crude umbilical cord serum. B) Two Dimension Electrophoresis Gel image of depleted umbilical cord serum by Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: regions of gel containing albumin, heavy and light chain of IgG before (panel A) and after depletion (panel B). Rectangle: used to highlight different protein patterns in the two groups, indicating a large number of protein spots that emerged in the depleted gel.
Mentions: As shown in Figure 2, albumin and IgG emerged in a large smeared protein pattern, masking proteins in a substantial area (Figure 2A, circles) on crude serum 2DE gels. Depletion of albumin and IgG by the PROTIA-Sigma kit improved the quality of 2DE gels in two ways: firstly, depletion of albumin and IgG resulted in improved separation within these regions of the gel and the emergence of several new spots (Figure 2B, circles). Additionally, the depletion of the high abundance proteins increased the load of low abundance proteins. Albumin and IgG represent about 80% of the total serum protein content and the PROTIA-Sigma kit was able to remove more than 90% of this albumin and IgG from the umbilical cord serum. Therefore, the loading capacity of low abundance proteins increased after protein depletion and many new protein spots emerged on the depleted serum 2DE gels (Figure 2B, Rectangle).

Bottom Line: Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots.During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels.Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, PR China. niejing@mail.sysu.edu.cn.

ABSTRACT

Background: High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum.

Results: The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins.

Conclusions: The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.

No MeSH data available.


Related in: MedlinePlus