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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

Comparison of depletion efficiency and repeatability of different kits on umbilical cord serum proteins samples by 1D-SDS-PAGE. Ten micrograms of crude, depleted or bound protein were loaded onto each lane and separated by 4-12% polyacrylamide gels. Proteins were visualized by Coomassie Blue staining. For examination of repeatability in umbilical cord serum depletion, the immunoaffinity columns (5185-Agilent and PROTIA-Sigma) were run three consecutive times with re-equilibration between depletion processes. However, for Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA), serum samples were tested in two independent columns since its depletion efficiency was low even with a new column. A) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. B) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in adult venous serum depletion. C) Multiple Affinity Removal System (5185, Agilent, Santa Clara, CA, USA) in umbilical cord serum depletion. D) Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. Lanes: M: Prestained protein marker (Fermentas; 250, 130; 95, 72, 55, 36, 28, 17); C: Crude serum; D: Depleted serum; B: Bound proteins; D1-3: Depleted umbilical cord serum proteins of the first, second and third depletion process. B1-3: Bound proteins eluted from the column after the first, second and third depletion process. Dp: Depleted Proteins pooled by the three depletion processes. Bp: Bound proteins pooled by the three depletion processes Closed arrows: Protein band indicating albumin; Open arrows: Protein band indicating IgG; *heavy chain, and **light chain.
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Figure 1: Comparison of depletion efficiency and repeatability of different kits on umbilical cord serum proteins samples by 1D-SDS-PAGE. Ten micrograms of crude, depleted or bound protein were loaded onto each lane and separated by 4-12% polyacrylamide gels. Proteins were visualized by Coomassie Blue staining. For examination of repeatability in umbilical cord serum depletion, the immunoaffinity columns (5185-Agilent and PROTIA-Sigma) were run three consecutive times with re-equilibration between depletion processes. However, for Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA), serum samples were tested in two independent columns since its depletion efficiency was low even with a new column. A) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. B) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in adult venous serum depletion. C) Multiple Affinity Removal System (5185, Agilent, Santa Clara, CA, USA) in umbilical cord serum depletion. D) Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. Lanes: M: Prestained protein marker (Fermentas; 250, 130; 95, 72, 55, 36, 28, 17); C: Crude serum; D: Depleted serum; B: Bound proteins; D1-3: Depleted umbilical cord serum proteins of the first, second and third depletion process. B1-3: Bound proteins eluted from the column after the first, second and third depletion process. Dp: Depleted Proteins pooled by the three depletion processes. Bp: Bound proteins pooled by the three depletion processes Closed arrows: Protein band indicating albumin; Open arrows: Protein band indicating IgG; *heavy chain, and **light chain.

Mentions: Immobilized dye based and immunoaffinity based methods are two major approaches in depletion of high abundance proteins. The Blue Albumin and IgG Depletion kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) is an immobilized dye based depletion kit which is highly efficient in depleting albumin and IgG from adult plasma/serum at a low cost. Therefore, we used the Blue Albumin and IgG Depletion kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) to measure the efficiency of albumin and IgG depletion from umbilical cord serum, using adult venous serum as control. In order to obtain the highest depletion efficiency, the lowest recommended amount of serum (25 μl) was loaded on the column. As shown in Figure 1A, after depletion by the PROTBA-Sigma kit, some of the highly abundant albumin and IgG bound to the column (Figure 1A, Lane B), however, a considerable amount still remained in the umbilical cord serum (Figure 1A, Lane D). On the contrary, the PROTBA-Sigma kit removed most of the albumin and IgG from adult venous serum (Figure 1B). This result suggests that the Blue Albumin and IgG Depletion kit could not deplete albumin and IgG effectively from umbilical cord serum. Since this immobilized dye based depletion kit did not show high efficiency, we then tested two immunoaffinity depletion kits using umbilical cord serum.


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)

Comparison of depletion efficiency and repeatability of different kits on umbilical cord serum proteins samples by 1D-SDS-PAGE. Ten micrograms of crude, depleted or bound protein were loaded onto each lane and separated by 4-12% polyacrylamide gels. Proteins were visualized by Coomassie Blue staining. For examination of repeatability in umbilical cord serum depletion, the immunoaffinity columns (5185-Agilent and PROTIA-Sigma) were run three consecutive times with re-equilibration between depletion processes. However, for Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA), serum samples were tested in two independent columns since its depletion efficiency was low even with a new column. A) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. B) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in adult venous serum depletion. C) Multiple Affinity Removal System (5185, Agilent, Santa Clara, CA, USA) in umbilical cord serum depletion. D) Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. Lanes: M: Prestained protein marker (Fermentas; 250, 130; 95, 72, 55, 36, 28, 17); C: Crude serum; D: Depleted serum; B: Bound proteins; D1-3: Depleted umbilical cord serum proteins of the first, second and third depletion process. B1-3: Bound proteins eluted from the column after the first, second and third depletion process. Dp: Depleted Proteins pooled by the three depletion processes. Bp: Bound proteins pooled by the three depletion processes Closed arrows: Protein band indicating albumin; Open arrows: Protein band indicating IgG; *heavy chain, and **light chain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Comparison of depletion efficiency and repeatability of different kits on umbilical cord serum proteins samples by 1D-SDS-PAGE. Ten micrograms of crude, depleted or bound protein were loaded onto each lane and separated by 4-12% polyacrylamide gels. Proteins were visualized by Coomassie Blue staining. For examination of repeatability in umbilical cord serum depletion, the immunoaffinity columns (5185-Agilent and PROTIA-Sigma) were run three consecutive times with re-equilibration between depletion processes. However, for Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA), serum samples were tested in two independent columns since its depletion efficiency was low even with a new column. A) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. B) Blue Albumin and IgG Depletion Kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) in adult venous serum depletion. C) Multiple Affinity Removal System (5185, Agilent, Santa Clara, CA, USA) in umbilical cord serum depletion. D) Immunoaffinity Albumin and IgG Depletion Kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA) in umbilical cord serum depletion. Lanes: M: Prestained protein marker (Fermentas; 250, 130; 95, 72, 55, 36, 28, 17); C: Crude serum; D: Depleted serum; B: Bound proteins; D1-3: Depleted umbilical cord serum proteins of the first, second and third depletion process. B1-3: Bound proteins eluted from the column after the first, second and third depletion process. Dp: Depleted Proteins pooled by the three depletion processes. Bp: Bound proteins pooled by the three depletion processes Closed arrows: Protein band indicating albumin; Open arrows: Protein band indicating IgG; *heavy chain, and **light chain.
Mentions: Immobilized dye based and immunoaffinity based methods are two major approaches in depletion of high abundance proteins. The Blue Albumin and IgG Depletion kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) is an immobilized dye based depletion kit which is highly efficient in depleting albumin and IgG from adult plasma/serum at a low cost. Therefore, we used the Blue Albumin and IgG Depletion kit (PROTBA, Sigma-Aldrich, Saint Louis, MO, USA) to measure the efficiency of albumin and IgG depletion from umbilical cord serum, using adult venous serum as control. In order to obtain the highest depletion efficiency, the lowest recommended amount of serum (25 μl) was loaded on the column. As shown in Figure 1A, after depletion by the PROTBA-Sigma kit, some of the highly abundant albumin and IgG bound to the column (Figure 1A, Lane B), however, a considerable amount still remained in the umbilical cord serum (Figure 1A, Lane D). On the contrary, the PROTBA-Sigma kit removed most of the albumin and IgG from adult venous serum (Figure 1B). This result suggests that the Blue Albumin and IgG Depletion kit could not deplete albumin and IgG effectively from umbilical cord serum. Since this immobilized dye based depletion kit did not show high efficiency, we then tested two immunoaffinity depletion kits using umbilical cord serum.

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