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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

Comparison of protein coverage in Hb-enriched fraction, Hb-depleted fraction, and unfractionated erythrocyte cytoplasm.A) Venn diagram of protein identifications directly proportional to the number of proteins in the overlap using one or two-fraction proteome analyses with corresponding histogram of protein identifications. B) Plots of protein intensity between Hb-enriched and Hb-depleted fractions in the two-fraction proteome, with black line denoting equal protein intensity in each fraction and red lines denoting a three-fold change.
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pone.0140036.g002: Comparison of protein coverage in Hb-enriched fraction, Hb-depleted fraction, and unfractionated erythrocyte cytoplasm.A) Venn diagram of protein identifications directly proportional to the number of proteins in the overlap using one or two-fraction proteome analyses with corresponding histogram of protein identifications. B) Plots of protein intensity between Hb-enriched and Hb-depleted fractions in the two-fraction proteome, with black line denoting equal protein intensity in each fraction and red lines denoting a three-fold change.

Mentions: Initial studies were performed to assess the specificity and utility of Hb-depletion using Ni-NTA columns and to evaluate the most efficient trade-off between depth of analysis and mass spectrometer analysis time using blood from a healthy donor. First, the total erythrocyte cytoplasm, Hb-depleted fraction, and hemoglobin eluate were analyzed in either one or two fractions following in-gel trypsin digestion using a 4 hour chromatographic gradient. Results from this analysis showed that as expected, far greater depth of analysis of the cytoplasm could be achieved when hemoglobin was removed, with identification of 688 proteins compared to 243 proteins identified in unfractionated cytosol (Fig 2A). An additional 50 proteins could be identified in the Hb-depleted cytoplasm when the cytosol was separated into two fractions on a short SDS gel. In comparison, only 443 proteins were identified with a two-fraction analysis of the entire cytosol due to the very high abundance of hemoglobin and limited amount of total peptides that can be loaded onto the LC column. The Hb-enriched fraction contained far fewer detectable proteins, with only 26 and 71 proteins identified in the single and two-fraction experiments, respectively (Fig 2B). Importantly, very few non-hemoglobin proteins were completely removed by the Ni-NTA column, as nearly all of the proteins identified in the Hb-enriched fraction were also identified in the Hb-depleted fraction. Comparison of the proteins that were identified in both the Hb-enriched and Hb-depleted fractions indicated that most proteins had a higher intensity in the Hb-depleted fraction, showing they were not enriched in the Hb-enriched fraction, suggesting only incidental retention on the column in a non-specific manner (Fig 2B). Furthermore, the three highest intensity proteins in the Hb-enriched fraction are all hemoglobin proteins, as expected. There are only four proteins that were found in the Hb-enriched fraction and were not detected in the Hb-depleted fraction (on X-axis in left panel of Fig 2B), as well as a handful of proteins that were higher intensity in the Hb-enriched fraction. This minor association of non-Hb proteins with the Ni-NTA column was somewhat variable. In fact, across four different Hb-depletion experiments, there were no proteins that were consistently found in the Hb-enriched fraction that were not detected in the Hb-depleted fractions. These results indicate that the depletion strategy is fairly specific for binding Hb and allows for improved depth of analysis of the erythrocyte cytoplasmic proteome.


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)

Comparison of protein coverage in Hb-enriched fraction, Hb-depleted fraction, and unfractionated erythrocyte cytoplasm.A) Venn diagram of protein identifications directly proportional to the number of proteins in the overlap using one or two-fraction proteome analyses with corresponding histogram of protein identifications. B) Plots of protein intensity between Hb-enriched and Hb-depleted fractions in the two-fraction proteome, with black line denoting equal protein intensity in each fraction and red lines denoting a three-fold change.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140036.g002: Comparison of protein coverage in Hb-enriched fraction, Hb-depleted fraction, and unfractionated erythrocyte cytoplasm.A) Venn diagram of protein identifications directly proportional to the number of proteins in the overlap using one or two-fraction proteome analyses with corresponding histogram of protein identifications. B) Plots of protein intensity between Hb-enriched and Hb-depleted fractions in the two-fraction proteome, with black line denoting equal protein intensity in each fraction and red lines denoting a three-fold change.
Mentions: Initial studies were performed to assess the specificity and utility of Hb-depletion using Ni-NTA columns and to evaluate the most efficient trade-off between depth of analysis and mass spectrometer analysis time using blood from a healthy donor. First, the total erythrocyte cytoplasm, Hb-depleted fraction, and hemoglobin eluate were analyzed in either one or two fractions following in-gel trypsin digestion using a 4 hour chromatographic gradient. Results from this analysis showed that as expected, far greater depth of analysis of the cytoplasm could be achieved when hemoglobin was removed, with identification of 688 proteins compared to 243 proteins identified in unfractionated cytosol (Fig 2A). An additional 50 proteins could be identified in the Hb-depleted cytoplasm when the cytosol was separated into two fractions on a short SDS gel. In comparison, only 443 proteins were identified with a two-fraction analysis of the entire cytosol due to the very high abundance of hemoglobin and limited amount of total peptides that can be loaded onto the LC column. The Hb-enriched fraction contained far fewer detectable proteins, with only 26 and 71 proteins identified in the single and two-fraction experiments, respectively (Fig 2B). Importantly, very few non-hemoglobin proteins were completely removed by the Ni-NTA column, as nearly all of the proteins identified in the Hb-enriched fraction were also identified in the Hb-depleted fraction. Comparison of the proteins that were identified in both the Hb-enriched and Hb-depleted fractions indicated that most proteins had a higher intensity in the Hb-depleted fraction, showing they were not enriched in the Hb-enriched fraction, suggesting only incidental retention on the column in a non-specific manner (Fig 2B). Furthermore, the three highest intensity proteins in the Hb-enriched fraction are all hemoglobin proteins, as expected. There are only four proteins that were found in the Hb-enriched fraction and were not detected in the Hb-depleted fraction (on X-axis in left panel of Fig 2B), as well as a handful of proteins that were higher intensity in the Hb-enriched fraction. This minor association of non-Hb proteins with the Ni-NTA column was somewhat variable. In fact, across four different Hb-depletion experiments, there were no proteins that were consistently found in the Hb-enriched fraction that were not detected in the Hb-depleted fractions. These results indicate that the depletion strategy is fairly specific for binding Hb and allows for improved depth of analysis of the erythrocyte cytoplasmic proteome.

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