Limits...
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

Principal component analysis and hierarchal clustering of DBA and healthy control samples.A) Principal component analysis shows a clear distinction between DBA and control cytosols with the exception of patient D6 which has a protein profile similar to that of healthy donors. B) Hierarchal clustering shows separation between most patients and controls.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608755&req=5

pone.0140036.g005: Principal component analysis and hierarchal clustering of DBA and healthy control samples.A) Principal component analysis shows a clear distinction between DBA and control cytosols with the exception of patient D6 which has a protein profile similar to that of healthy donors. B) Hierarchal clustering shows separation between most patients and controls.

Mentions: Label-free quantitation and data normalization was performed using the MaxQuant software package with matching between runs to yield a final list of 1052 proteins (S2 Table). Principal component analysis and hierarchal clustering, using the intensities of all 1052 proteins, showed good separation between most DBA patients and controls, indicating an inherent difference between the proteomes in each cohort. The most obvious exceptions were the proteomes of patient D6, which clustered with the healthy donor proteomes (Fig 5). This similarity was consistent with the patient’s complete blood counts, which were well within the normal range, indicating that the patient had undergone spontaneous remission (Table 1). Similarly, clinical blood parameters for patient D4 were mostly within the normal range, although Hb values were at the lower boundary, and principal component analysis showed this patient’s proteome was closer to the controls than the remaining DBA patients. These observations suggest that with the recovery to normal blood count values, DBA patients’ erythrocyte cytoplasm proteomes resemble normal proteomes. As patient D6 appeared to be normal based on both clinical parameters and total proteome, data from this patient were not further considered when comparing DBA and control proteomes. Interestingly, all controls were tightly clustered regardless of gender or age, suggesting that all controls could be treated as a single homogeneous group. Not surprisingly, the DBA patient proteomes were more heterogeneous, although the two different blood draws from the same patient were usually closely clustered in the principle component analysis plot and hierarchal cluster analysis (Fig 5). Due to the small variation seen between the male and female control donors in the principal component analysis, all subsequent comparisons were performed between all DBA patients (excluding patient D6) versus all controls.


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)

Principal component analysis and hierarchal clustering of DBA and healthy control samples.A) Principal component analysis shows a clear distinction between DBA and control cytosols with the exception of patient D6 which has a protein profile similar to that of healthy donors. B) Hierarchal clustering shows separation between most patients and controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140036.g005: Principal component analysis and hierarchal clustering of DBA and healthy control samples.A) Principal component analysis shows a clear distinction between DBA and control cytosols with the exception of patient D6 which has a protein profile similar to that of healthy donors. B) Hierarchal clustering shows separation between most patients and controls.
Mentions: Label-free quantitation and data normalization was performed using the MaxQuant software package with matching between runs to yield a final list of 1052 proteins (S2 Table). Principal component analysis and hierarchal clustering, using the intensities of all 1052 proteins, showed good separation between most DBA patients and controls, indicating an inherent difference between the proteomes in each cohort. The most obvious exceptions were the proteomes of patient D6, which clustered with the healthy donor proteomes (Fig 5). This similarity was consistent with the patient’s complete blood counts, which were well within the normal range, indicating that the patient had undergone spontaneous remission (Table 1). Similarly, clinical blood parameters for patient D4 were mostly within the normal range, although Hb values were at the lower boundary, and principal component analysis showed this patient’s proteome was closer to the controls than the remaining DBA patients. These observations suggest that with the recovery to normal blood count values, DBA patients’ erythrocyte cytoplasm proteomes resemble normal proteomes. As patient D6 appeared to be normal based on both clinical parameters and total proteome, data from this patient were not further considered when comparing DBA and control proteomes. Interestingly, all controls were tightly clustered regardless of gender or age, suggesting that all controls could be treated as a single homogeneous group. Not surprisingly, the DBA patient proteomes were more heterogeneous, although the two different blood draws from the same patient were usually closely clustered in the principle component analysis plot and hierarchal cluster analysis (Fig 5). Due to the small variation seen between the male and female control donors in the principal component analysis, all subsequent comparisons were performed between all DBA patients (excluding patient D6) versus all controls.

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