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In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Pesciotta EN, Lam HS, Kossenkov A, Ge J, Showe LC, Mason PJ, Bessler M, Speicher DW - PLoS ONE (2015)

Bottom Line: Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors.Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively].These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd Philadelphia, PA, 19104, United States of America; Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce St. Philadelphia, PA, 19104, United States of America.

ABSTRACT
Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

No MeSH data available.


Related in: MedlinePlus

Schematic of erythrocyte purification and cytosolic protein enrichment for label-free proteomic analysis.
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pone.0140036.g001: Schematic of erythrocyte purification and cytosolic protein enrichment for label-free proteomic analysis.

Mentions: As discussed above, proteomics analysis of erythrocyte cytosol is complicated by the very high abundance of hemoglobin. It was therefore necessary to develop an analytical scheme where small volumes of patient blood samples could be analyzed in depth, while simultaneously keeping mass spectrometer analysis time within realistic limits. Therefore, we evaluated the utility of Ni-NTA columns for effective, reproducible depletion of hemoglobin using small blood volumes. We then evaluated the effects of varying degrees of fractionation and LC-MS/MS analysis parameters on depth of analysis of the Hb-depleted cytosolic erythrocyte proteome. Approximately 3–8 mL of peripheral blood in EDTA tubes were obtained from patients and healthy individuals with informed written consent according to the Declaration of Helsinki. Erythrocyte purification was performed as previously described [17] and included leukocyte depletion and elimination of other contaminating cell types by repeated centrifugations and buffy coat removal. Purified erythrocytes were then lysed and separated into membranes (erythrocyte ghosts) and cytoplasm fractions prior to in-gel trypsin digestion and analysis by LC-MS/MS (Fig 1).


In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Pesciotta EN, Lam HS, Kossenkov A, Ge J, Showe LC, Mason PJ, Bessler M, Speicher DW - PLoS ONE (2015)

Schematic of erythrocyte purification and cytosolic protein enrichment for label-free proteomic analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608755&req=5

pone.0140036.g001: Schematic of erythrocyte purification and cytosolic protein enrichment for label-free proteomic analysis.
Mentions: As discussed above, proteomics analysis of erythrocyte cytosol is complicated by the very high abundance of hemoglobin. It was therefore necessary to develop an analytical scheme where small volumes of patient blood samples could be analyzed in depth, while simultaneously keeping mass spectrometer analysis time within realistic limits. Therefore, we evaluated the utility of Ni-NTA columns for effective, reproducible depletion of hemoglobin using small blood volumes. We then evaluated the effects of varying degrees of fractionation and LC-MS/MS analysis parameters on depth of analysis of the Hb-depleted cytosolic erythrocyte proteome. Approximately 3–8 mL of peripheral blood in EDTA tubes were obtained from patients and healthy individuals with informed written consent according to the Declaration of Helsinki. Erythrocyte purification was performed as previously described [17] and included leukocyte depletion and elimination of other contaminating cell types by repeated centrifugations and buffy coat removal. Purified erythrocytes were then lysed and separated into membranes (erythrocyte ghosts) and cytoplasm fractions prior to in-gel trypsin digestion and analysis by LC-MS/MS (Fig 1).

Bottom Line: Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors.Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively].These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd Philadelphia, PA, 19104, United States of America; Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce St. Philadelphia, PA, 19104, United States of America.

ABSTRACT
Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

No MeSH data available.


Related in: MedlinePlus