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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

Triplicate Hb-depletions show high reproducibility.A) Number of protein identifications in Hb-enriched fractions and Hb-depleted fractions from triplicate depletions of a single erythrocyte cytosol preparation. B) Number of peptide identifications in these triplicate Hb depletions. C) Histogram showing coefficient of variation in protein intensity across triplicate analyses of Hb-enriched fractions with embedded Venn diagram showing protein overlap between these three datasets. D) Histogram of the number of proteins with corresponding coefficient of variation for triplicate Hb-depleted fractions with Venn diagram showing almost complete overlap of over 750 proteins.
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pone.0140036.g004: Triplicate Hb-depletions show high reproducibility.A) Number of protein identifications in Hb-enriched fractions and Hb-depleted fractions from triplicate depletions of a single erythrocyte cytosol preparation. B) Number of peptide identifications in these triplicate Hb depletions. C) Histogram showing coefficient of variation in protein intensity across triplicate analyses of Hb-enriched fractions with embedded Venn diagram showing protein overlap between these three datasets. D) Histogram of the number of proteins with corresponding coefficient of variation for triplicate Hb-depleted fractions with Venn diagram showing almost complete overlap of over 750 proteins.

Mentions: To test the reproducibility of Hb-depletion with Ni-NTA columns, triplicate Hb-depletion was performed using three aliquots of the same erythrocyte cytosol preparation and three Ni-NTA depletion columns. Each Hb-depleted fraction and Hb-eluate was separated into two fractions on short SDS gels and resulting tryptic digests were analyzed by LC-MS/MS. Results from this experiment confirmed that Hb-depletion with Ni-NTA columns is quite reproducible with similar protein and peptide identifications across the triplicate datasets (Fig 4A and 4B). The reproducibility of protein intensities across triplicates was also excellent with coefficients of variation below 5% for most proteins in both the Hb-enriched fractions and Hb-depleted fractions (Fig 4C and 4D). The overlap of protein identifications in triplicate analysis of the Hb-enriched fractions and Hb-depleted fractions also was highly reproducible, especially for the Hb-depleted fractions that have almost complete overlap in protein identifications (Fig 4C and 4D). The larger variation of protein identifications in the Hb-enriched fractions is due to increased variability in detecting lower abundant proteins in the presence of a few very high abundance proteins. Specifically, 88% of the MS/MS spectra that resulted in peptide identifications in the Hb-enriched fraction were for hemoglobin proteins. These results indicate that Hb-enriched depletion with Ni-NTA columns is a very reproducible and reliable way to increase protein identifications of erythrocyte cytoplasmic proteins in a manner that is highly amenable to label-free quantitation.


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)

Triplicate Hb-depletions show high reproducibility.A) Number of protein identifications in Hb-enriched fractions and Hb-depleted fractions from triplicate depletions of a single erythrocyte cytosol preparation. B) Number of peptide identifications in these triplicate Hb depletions. C) Histogram showing coefficient of variation in protein intensity across triplicate analyses of Hb-enriched fractions with embedded Venn diagram showing protein overlap between these three datasets. D) Histogram of the number of proteins with corresponding coefficient of variation for triplicate Hb-depleted fractions with Venn diagram showing almost complete overlap of over 750 proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140036.g004: Triplicate Hb-depletions show high reproducibility.A) Number of protein identifications in Hb-enriched fractions and Hb-depleted fractions from triplicate depletions of a single erythrocyte cytosol preparation. B) Number of peptide identifications in these triplicate Hb depletions. C) Histogram showing coefficient of variation in protein intensity across triplicate analyses of Hb-enriched fractions with embedded Venn diagram showing protein overlap between these three datasets. D) Histogram of the number of proteins with corresponding coefficient of variation for triplicate Hb-depleted fractions with Venn diagram showing almost complete overlap of over 750 proteins.
Mentions: To test the reproducibility of Hb-depletion with Ni-NTA columns, triplicate Hb-depletion was performed using three aliquots of the same erythrocyte cytosol preparation and three Ni-NTA depletion columns. Each Hb-depleted fraction and Hb-eluate was separated into two fractions on short SDS gels and resulting tryptic digests were analyzed by LC-MS/MS. Results from this experiment confirmed that Hb-depletion with Ni-NTA columns is quite reproducible with similar protein and peptide identifications across the triplicate datasets (Fig 4A and 4B). The reproducibility of protein intensities across triplicates was also excellent with coefficients of variation below 5% for most proteins in both the Hb-enriched fractions and Hb-depleted fractions (Fig 4C and 4D). The overlap of protein identifications in triplicate analysis of the Hb-enriched fractions and Hb-depleted fractions also was highly reproducible, especially for the Hb-depleted fractions that have almost complete overlap in protein identifications (Fig 4C and 4D). The larger variation of protein identifications in the Hb-enriched fractions is due to increased variability in detecting lower abundant proteins in the presence of a few very high abundance proteins. Specifically, 88% of the MS/MS spectra that resulted in peptide identifications in the Hb-enriched fraction were for hemoglobin proteins. These results indicate that Hb-enriched depletion with Ni-NTA columns is a very reproducible and reliable way to increase protein identifications of erythrocyte cytoplasmic proteins in a manner that is highly amenable to label-free quantitation.

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