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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

Identification of protein spots unique to depleted umbilical cord serum gels by the Immunoaffinity Albumin and IgG Depletion kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: Ten spots unique to the depleted gels were selected for MALDI-TOF/TOF MS identification. Gel image is the same as in Fig 2B. The numbers correspond to the protein numbers in Table 2.
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Figure 3: Identification of protein spots unique to depleted umbilical cord serum gels by the Immunoaffinity Albumin and IgG Depletion kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: Ten spots unique to the depleted gels were selected for MALDI-TOF/TOF MS identification. Gel image is the same as in Fig 2B. The numbers correspond to the protein numbers in Table 2.

Mentions: Next, ten unique spots from the depleted gels were randomly chosen for identification by MALDI-TOF/TOF MS, with selected protein spots indicated in Figure 3 (Gel image in Figure 3 is the same as in Figure 2B). Eight of ten spots were successfully identified by MS, with identifications determined according to the highest Mascot MS/MS score (Table 2). The majority of the spots, including Zinc-alpha-2-glycoprotein, Hematopoietic lineage cell-specific protein, Tubulin alpha chain-like 3, Apolipoprotein E, Tropomyosin beta chain, Sorcin, Tetranectin, were low abundance proteins. These protein spots could not have been directly detected in the crude umbilical cord serum 2DE gels due to interference by high abundance proteins, but became apparent in the depleted serum 2DE gels.


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)

Identification of protein spots unique to depleted umbilical cord serum gels by the Immunoaffinity Albumin and IgG Depletion kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: Ten spots unique to the depleted gels were selected for MALDI-TOF/TOF MS identification. Gel image is the same as in Fig 2B. The numbers correspond to the protein numbers in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Identification of protein spots unique to depleted umbilical cord serum gels by the Immunoaffinity Albumin and IgG Depletion kit (PROTIA, Sigma-Aldrich, Saint Louis, MO, USA). Circles: Ten spots unique to the depleted gels were selected for MALDI-TOF/TOF MS identification. Gel image is the same as in Fig 2B. The numbers correspond to the protein numbers in Table 2.
Mentions: Next, ten unique spots from the depleted gels were randomly chosen for identification by MALDI-TOF/TOF MS, with selected protein spots indicated in Figure 3 (Gel image in Figure 3 is the same as in Figure 2B). Eight of ten spots were successfully identified by MS, with identifications determined according to the highest Mascot MS/MS score (Table 2). The majority of the spots, including Zinc-alpha-2-glycoprotein, Hematopoietic lineage cell-specific protein, Tubulin alpha chain-like 3, Apolipoprotein E, Tropomyosin beta chain, Sorcin, Tetranectin, were low abundance proteins. These protein spots could not have been directly detected in the crude umbilical cord serum 2DE gels due to interference by high abundance proteins, but became apparent in the depleted serum 2DE gels.

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