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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

Effects of gradient length and extent of proteome fractions on protein and peptide identifications using Hb-depleted samples.Replicate proteomes were analyzed using a 4-hour gradient to analyze an unfractionated sample or a sample fractionated into two or four fractions. The unfractionated sample also was analyzed using duplicate 4-hour gradients or a single 8-hour gradient. A) Protein identifications across Hb-depleted datasets. B) Peptide identifications across Hb-depleted datasets.
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pone.0140036.g003: Effects of gradient length and extent of proteome fractions on protein and peptide identifications using Hb-depleted samples.Replicate proteomes were analyzed using a 4-hour gradient to analyze an unfractionated sample or a sample fractionated into two or four fractions. The unfractionated sample also was analyzed using duplicate 4-hour gradients or a single 8-hour gradient. A) Protein identifications across Hb-depleted datasets. B) Peptide identifications across Hb-depleted datasets.

Mentions: Next we ran a series of experiments to determine the optimal trade-off between total instrument time per sample and depth of analysis when analyzing the Hb-depleted sample using blood from a healthy donor. Length of gradient and number of fractions per proteome were evaluated. In this experiment, analysis of a single fraction proteome using a 4 hour LC-MS/MS run provided 615 proteins. However, a two-fraction proteome was substantially superior with 756 proteins identified, while a four-fraction analysis identified 798 proteins (Fig 3A). Duplicate analysis of a single fraction proteome used the same amount of mass spectrometer time as analysis of a two fraction proteome but less depth of analysis than the two fraction proteome, with only 696 proteins identified. We also tested a single-fraction proteome using an 8-hour chromatographic gradient, which did not improve performance with only 635 proteins identified. While the most peptides were identified in the four-fraction analysis (Fig 3B), this increased depth of analysis only contributed to 42 more protein identifications, which was regarded as insufficient to justify the doubled mass spectrometer time. Therefore, based on these experiments, the best trade-off in depth of analysis and instrument time was regarded as analysis of Hb-depleted cytosol separated into two-fractions and analyzed using 4-hour LC-MS/MS runs.


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)

Effects of gradient length and extent of proteome fractions on protein and peptide identifications using Hb-depleted samples.Replicate proteomes were analyzed using a 4-hour gradient to analyze an unfractionated sample or a sample fractionated into two or four fractions. The unfractionated sample also was analyzed using duplicate 4-hour gradients or a single 8-hour gradient. A) Protein identifications across Hb-depleted datasets. B) Peptide identifications across Hb-depleted datasets.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140036.g003: Effects of gradient length and extent of proteome fractions on protein and peptide identifications using Hb-depleted samples.Replicate proteomes were analyzed using a 4-hour gradient to analyze an unfractionated sample or a sample fractionated into two or four fractions. The unfractionated sample also was analyzed using duplicate 4-hour gradients or a single 8-hour gradient. A) Protein identifications across Hb-depleted datasets. B) Peptide identifications across Hb-depleted datasets.
Mentions: Next we ran a series of experiments to determine the optimal trade-off between total instrument time per sample and depth of analysis when analyzing the Hb-depleted sample using blood from a healthy donor. Length of gradient and number of fractions per proteome were evaluated. In this experiment, analysis of a single fraction proteome using a 4 hour LC-MS/MS run provided 615 proteins. However, a two-fraction proteome was substantially superior with 756 proteins identified, while a four-fraction analysis identified 798 proteins (Fig 3A). Duplicate analysis of a single fraction proteome used the same amount of mass spectrometer time as analysis of a two fraction proteome but less depth of analysis than the two fraction proteome, with only 696 proteins identified. We also tested a single-fraction proteome using an 8-hour chromatographic gradient, which did not improve performance with only 635 proteins identified. While the most peptides were identified in the four-fraction analysis (Fig 3B), this increased depth of analysis only contributed to 42 more protein identifications, which was regarded as insufficient to justify the doubled mass spectrometer time. Therefore, based on these experiments, the best trade-off in depth of analysis and instrument time was regarded as analysis of Hb-depleted cytosol separated into two-fractions and analyzed using 4-hour LC-MS/MS runs.

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