Conceived and designed the experiments: KvL JSF TMS. Performed the experiments: KvL TMS KS JSF. Analyzed the data: KvL TMS KS XX JSF. Wrote the paper: KvL JSF.
The authors have declared that no competing interests exist.
Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis.
Red blood cells (RBC), the most abundant cell type in the human body, are highly specialized structurally and functionally to supply oxygen to tissues via the circulatory system. Erythrocyte development begins with marrow progenitors under the influence of lineage specific hematopoietic growth factors, with erythropoietin being the critical growth factor governing RBC production. Marrow RBC development progresses until immature RBC extrude their nuclei and exit the bone marrow as newly formed reticulocytes
Hereditary non-spherocytic hemolytic anemias (HNSHA) are a heterogeneous group of RBC enzymatic disorders with PK and G6PD deficiencies being the most common lesions
Normal Range | HA09 |
HA19 | HA21 | HA24 |
|
Age (years) | 9.75 | 2 | 16.25 | 0.5 | |
Sex | m | f | f | f | |
|
|||||
Hemoglobin (Hb)/(g/dl) | m: 13–18f: 12–16 | 8.9 | 7.3 | 12.7 | 7.4 |
Hematocrit (HTC)/(%) | m: 45–52f: 37–48 | 26.9 | 23 | 34 | 22.3 |
Red cell count (x 1012/l) | m: 4.2–5.9f: 3.8–5.5 | 3.09 | 3.1 | 3.57 | 2.49 |
Reticulocytes (%) | m: 0.92–2.71f: 0.61–2.2 | 3.45 | 11.6 | 18.4 | 6.21 |
Unstable Hemoglobin | 0 = negative1 = positive | 0 | 0 | 0 | 0 |
|
|||||
Pyruvate Kinase | 11.1– 18.9 | 13 | 23.2 | 11.8 | 5.3 |
G6PD | 7.9 – 16.3 | 9.8 | 25.9 | 14.9 | ND |
Hexokinase | 1.02– 2.54 | 1.62 | 5.17 | 1.9 | 3.45 |
GPI | 38.8– 82.2 | 49 | 86.3 | 60.6 | ND |
TPI | 1317– 2905 | ND | 1602 | 1387 | ND |
Glutathione Peroxidase | 21.3– 40.3 | 30.2 | ND | 44 | ND |
Reduced Glutathione | 4.5 – 8.7 | 6.14 | 7.78 | ND | 8.19 |
blood transfusion prior to drawing blood: HA09 42 days, HA24 37 days.
m: male.
f: female.
UI: International Unit.
G6PD: Glucose -6- Phosphate Dehydrogenase.
TPI: Triose Phosphate Isomerase.
GPI: Glucose Phosphate Isomerase.
ND: No Data.
The complete panel of enzyme assays performed on all samples in this study including controls can be found in Supplementary
HA19 is a 2-year old female who presented with chronic, hemolytic anemia accompanied by splenomegaly, leucopenia and thrombocytopenia. Initial work up was negative for common enzyme deficiencies (G6PD and PK), hemoglobinopathy or membrane defects, and there was no family history of chronic anemia. Lab values concurrent with sampling for this study include hematocrit of 23%, hemoglobin of 7.3, RBC count of 3.1x1012/l and reticulocytes of 11.6%. Evaluation in our lab revealed no abnormalities in G6PD, HK, PK, GOT, GPI, GPx, triose phosphate isomerase (TPI) or phosphoglycerate kinase (PGK), with GSH in the normal range (
HA21 is a 15-year-old female with mild chronic anemia of unknown cause and a history of a previous acute hemolytic event. Prior evaluation showed no evidence of common enzyme deficiency (G6PD, PK, HK, GPI, TPI and GPx reported as normal), hemoglobinopathy or membrane defect, with normal osmotic fragility. Lab values concurrent with sampling for this study include hematocrit of 34%, hemoglobin of 12.7 g/dl, RBC count of 3.57x1012/l and reticulocytes of 18%. Enzyme analyses in our laboratory were negative for deficiencies, but showed elevated GPx in both the patient and her mother. In the absence of an identified defect, proteomic analysis was performed comparing HA21 (patient), her mother and two unrelated control samples.
HA24 is a 6-month old female with severe, chronic anemia requiring transfusion approximately every 5 weeks. As with HA09, a sample for analysis was obtained at the nadir, just prior to transfusion for analysis. At the time of sampling, hematocrit was 22.3%, hemoglobin 7.4 g/dl, RBC count 2.49x1012/l and reticulocytes 6.21%. Prior workup for common enzyme deficiencies, hemoglobinopathy and membrane defects were negative. HK activity and GSH levels, were in the normal range. Despite a report of normal PK activity as part of the patient’s original evaluation, enzyme analysis in our laboratory was suggestive of PK deficiency, particularly considering the patient’s reticulocyte count and history of transfusion, with patient, mother and father all showing decreased activity (
Spots | HA09 | HA19 | HA21 | HA24 |
Total number | 963 | 656 | 956 | 1042 |
Differentially expressed (ANOVA≤0.05) | 488 | 411 | 687 | 580 |
Excluded | 291 | 256 | 477 | 471 |
Included | 197 | 155 | 210 | 109 |
Picked | 48 | 48 | 99 | 48 |
w/o ID | 7 | 1 | 1 | 2 |
Proteins identified | 53 | 63 | 145 | 72 |
Total number: Number of spots detected by Same Spots Software after filtering (area size and minimum volume).
Differentially expressed: Number of spots where difference of expression (normalized volume) of two samples in the experimental set meet the statistical criteria of ANOVA≤0.05.
Excluded: Number of spots that were excluded.
Included: Number of “Differentially expressed” minus “Excluded”.
Picked: Number of spots picked and trypsinized for mass spec analysis.
w/o ID: Number of spots where no peptide could be identified.
Proteins Identified: Total number of Proteins identified in all spots picked in experimental set. Note that more than one protein can be identified in a single picked spot.
As an overall data quality metric, Principal Component Analysis (PCA,
Dot plot of all spots included in analysis (see
Assessment of expression profiles of HA09 showed down-regulation of proteins to be more common than up regulation, with more than twice as many spots showing lower expression in the patient (
Of all spots included in the analyses (see
ID | Spot rank | Fold change | Gene | Protein | |
HA09 | |||||
82 | - 2.63 | G6PD | Glucose-6-phosphate 1-dehydrogenase | ||
CAP1 | Adenylyl cyclase-associated protein 1 | ||||
160 | - 1.93 | G6PD | Glucose-6-phosphate 1-dehydrogenase | ||
232 | - 1.73 | PA2G4 | Proliferation-associated protein 2G4 | ||
307 | - 1.61 | PA2G4 | Proliferation-associated protein 2G4 | ||
HA19 | |||||
89 | - 2.14 | HSPA8 | Isoform 1 of Heat shock cognate 71kD | ||
94 | - 1.92 | LOC44091 | Similar to 14-3-3 protein epsilon | ||
231 | +1.68 | PITHD1 | PITH domain-containing protein 1 | ||
HA21 | |||||
28 | +3.27 | EIF2S3 | Eukaryotic translation initiation factor 2, subunit 3 | ||
61 | +2.58 | PSMB4 | Proteasome subunit beta type-4 (precursor) | ||
67 | +3.12 | ACTR1A | Alpha-centractin | ||
162 | +4.04 | VCP | Valosin-containing protein | ||
200 | +2.01 | VCP | Valosin-containing protein | ||
204 | -1.93 | SOD1 | Superoxide dismutase 1, soluble | ||
258 | +1.89 | HDHD2 | Haloacid dehalogenase-like hydrolase domain containing 2 | ||
HA24 | |||||
4 | +4.57 | EEF2 | Elongation factor 2 | ||
13 | +4.33 | RPSA | Ribosomal protein SA | ||
14 | +3.03 | XPO7 | Exportin 7 | ||
36 | +2.28 | BPGM | Bisphosphoglycerate mutase | ||
43 | +2.28 | BPGM | Bisphosphoglycerate mutase | ||
55 | +1.86 | XPO7 | Exportin 7 | ||
97 | -1.76 | CCT8 | Chaperonin containing TCP1, subunit 8 | ||
132 | +1.60 | ADSL | Isoform 1 of Adenylosuccinate lyase | ||
159 | +1.55 | PRDX2 | Peroxiredoxin 2 | ||
167 | +1.61 | PGAM1 | Phosphoglycerate mutase 1 |
ID: Name of sample set (HA09, HA19, HA21, HA24).
Spot rank: Rank of spot as assigned by Same Spots Software depending on fold change (normalized volume) comparing the highest to lowest sample,
Fold Change: Comparison of normalized volume in Patient sample with average of Control and Standard sample.
Gene: HGNC Symbol for coding human gene.
Protein: HGNC Symbol for protein identified.
By superimposing images of Coomassie stained gels from all four experiments including the positions of the picked spots, it was possible to identify differentially expressed spots that were picked in more than one experiment. Across all gels, protein identification was overlapping with the same major protein species present in all cases, and additional peptides from minor species being variably present. As an initial method of protein ID verification the location of the excised spot was compared with the molecular weight (kD) and presumed pI of the identified protein(s). When the discrepancy was > 20% of theoretical molecular mass or > 1 pI unit, it was often found that peptides were derived from the previously picked spot (representing contamination at the level of the spot picking head) and could thus be excluded from further analysis. In some instances we found multiple peptides mapping to a protein larger than the spot location could only be aligned to one part of that protein, suggesting that the protein fragmented prior to gel separation, either as an artifact of processing or a physiologic cleavage prior to sample processing. A representative Coomassie stained 2-D gel is shown in
Proteins that were expressed at higher levels in patients are represented by green ovals, while down-regulated proteins are represented by red ovals.
XPO7 is a member of the importin-beta superfamily of nuclear transport receptors thought to cycle between the cytoplasm and the nucleus
Graphs shows expression levels of Exportin 7 (A), Fumarate Hydratase (B) and Purine Nucleoside Phosphorylase (C) in normalized volume for both replicates run per indicated sample. Included are all spots in which the indicated proteins were identified as the predominant protein. D: Images of gel sections (three for HA19, left panel; four images for HA24, right panel) were merged to show the area where spots were excised. Blue lines enclose spot areas.
The tricarboxylic acid cycle (TCA) enzyme fumarate hydratase, which normally localizes to mitochondria, was unexpectedly found to be present in RBC cytosol. A possible explanation for the presence of this enzyme would be as a component of residual mitochondria in circulating reticulocytes. In patients HA19 and HA21, this explanation appears incorrect, as fumarate hydratase protein was reduced more than 2 fold in these patients compared to controls (
Purine nucleoside phosphorylase (PNP) was identified in several spots in all experiments. PNP deficiency is a rare autosomal recessive disorder characterized by autoimmunity that may include hemolytic anemia
Abundance of specific cytoskeletal proteins differed in all four patients (Supplementary
Two proteins thought to be involved in ribosome assembly and/or stability were differentially expressed in all four patients. Proliferation-associated protein 2G4 (P2G4) is part of a pre-ribosomal ribonucloeprotein complex and has been implicated in growth regulation in human fibroblasts
Chaperones were also differentially expressed in all patients (
Profiles for HA09 are depicted in (A), HA19 (B), HA21(C) and HA24 (D). Graphs show expression levels in normalized volume for both replicates run per indicated sample. Red lines represent expression patterns of T-complex protein subunits; Heatshock proteins are in blue.
A prominent common pattern observed in all patient samples was reduced abundance of multiple proteasome subunits as well as reduced expression of additional proteins involved in protein degradation (
Graphs show expression levels in normalized volume for both replicates run per indicated sample (A: HA09, B: HA19, C: HA21 and D: HA24). Two spot expression patterns in HA21 differ from the others: PSMB4 is expressed at reduced levels in both patient and her mother (blue line) and PSMC5 is expressed at increased levels in the patient (green line).
In order to focus upon protein expression changes that could play a role in etiology of HNSHA, we searched for spot patterns where the volume measured in the patient sample was most distinct from the control samples, and one or both parents showed intermediate expression. We reasoned that such a pattern might be indicative of recessive inheritance of a lesion for which both parents were heterozygous (
Shown are Graphs (in normalized volume) for both replicates run per indicated sample in experimental set HA09 (A), HA19 (B), HA21 (C, left panel: patient lower expressing, right panel: patient higher expressing) and HA24 (D, left panel: patient lower expressing, right panel: patient higher expressing). Numbers (#) refer to Spot ranks in
In HA09 (
In HA24, CCT8 (T-complex chaperone subunit, #97) was identified as a protein with low expression in both the patient and mother (
Known causes of hereditary non-spherocytic hemolytic anemia (HNSHA) include lesions affecting enzymes involved in glycolysis, the pentose phosphate pathway, gluthathione metabolism, and nucleotide homeostasis
Each sample was evaluated on 2 separate gels using a dye reversal approach
An obvious difference between control and HNSHA RBC samples is the increased reticulocyte count in patient samples. HA09 and HA24 differ from HA19 and HA21 in that they are both transfusion dependent–indicating their ability to produce RBC is more significantly impaired. Because reticulocytosis by itself will increase the abundance of protein species that are normally lost during erythroid maturation, differential expression of mitochondrial proteins, components of protein synthetic machinery and heme biosynthetic pathway, as elucidated in a recent analysis of a mouse hemolytic anemia model
Exportin 7 (XPO7), a protein involved in shuttling macromolecules across the nuclear envelope
RPSA, a protein important for ribosome assembly and stability of the 40S ribosome, was increased in patients HA19, HA21 and HA24. RPSA, also named laminin receptor 1
Eukaryotic elongation factor 2 (eEF2), a protein that mediates mRNA translation at ribosomes, was increased in patients HA21 and HA24 (
Patient HA09 is also transfusion dependent, and the combination of low endogenous RBC production in a background of normal, transfused RBC likely explains why this sample had fewer proteins with significantly increased expression. This supports the conjecture that the observed increase in abundance of many proteins in HA patient samples is a marker of the relative increase in reticulocyte count.
Endogenous protein quality control is a critical process, especially for a cell such as the erythrocyte with limited capacity to replace or repair damaged proteins. The degradation of damaged or defective proteins is an important homeostatic mechanism to avoid protein aggregation, membrane damage and cell death
Reduced expression of multiple subunits of the 26S proteasome as well as other proteins of the ubiquitin-proteasome system was found in all HA patient samples (
During maturation from reticulocyte to erythrocyte extensive reorganization of the cytoskeleton and membrane occur in order to maximize cell malleability and shear resistance. Microfilamants and microtubles are essential for cell motility and the extrusion of the nucleus
Fumarate hydratase (FH) is a tricarboxylic cycle (TCA) enzyme with reduced expression in patients HA19 and HA21 (
HA19 and HA21 also showed reduction in expression of purine nucleoside phosphorylase (PNP,
Although outside screening had been negative, we suspected patient HA24 could be PK deficient based upon borderline low PK activity and below control values in both parents when assayed in our laboratory (
Lack of sensitivity of the DIGE approach reflects a number of technical choices/challenges in carrying out this study. First, we limited analysis to cytosolic proteins, excluding the membrane fraction because of poor resolution of membrane proteins on 2D gels. Second, we ran the cytosolic fractions over DEAE in order to remove hemoglobin and thus increase sensitivity for lower abundance cytosolic proteins. It is likely that some cytosolic proteins were removed in this step along with hemoglobin. Finally, proteins with extreme isoelectric points (below 4.0 and above 8.5) were outside the range of resolution of our 2D gels. While the above comments relate to technical limitations of 2-D DIGE, there are additional limitations inherent in experimental design when comparing single clinical samples against related and unrelated controls. Here, in order to allow meaningful statistical comparisons, each sample was run twice using a dye-reversal strategy that also corrects for biases in the protein labeling or fluorescence detection steps. While this approach controls for experimental variations, when differential protein expression between patients and controls is observed, we cannot discriminate between anemia-related differential protein expression and differences in expression that represent a polymorphism between the patient and controls that is not disease associated.
In addition to gel separation problems, we encountered single proteins that ran in several spots next to each other, raising problems for relative quantification. Examples include G6PD, BPGM, eEF2, XPO 7 (
Evaluation and treatment of patients with hereditary nonspherocytic hemolytic anemia continues to be a challenge. By using a proteomic approach to evaluate four patients with this condition we found a common pattern of altered expression of proteins involved in protein quality control and degradation, raising the possibility that interventions to increase chaperone activity may benefit some patients by diminishing accumulation of damaged or misfolded proteins. We also identified several patient-specific alterations in protein expression that require further investigation (
Peripheral blood samples from HA patients, unaffected family members and controls were collected after obtaining informed consent at the referring institution, using consent documentation approved by the Scripps Research Institute IRB (La Jolla, CA) that specifically approved this study. Because each HA patient was a minor, consent documentation was signed by parent/guardian, and, when age-appropriate, by participating patient. Control samples obtained from family members utilized the same informed consent documentation and procedure. Non-familial control samples were obtained from the Scripps Normal Blood Donor pool, again after obtaining informed consent using documentation of the Scripps Research Institute IRB that specifically approved this study. 7–10cc of whole blood was collected into EDTA tubes and shipped overnight on wet ice.
White cells were removed from whole blood by passage over microcrystalline cellulose columns and washed 3 times in 0.9% saline solution. RBC were frozen at -80°C prior to fractionation for proteomic analysis. Reticulocyte count was assessed using BD Retic-Count™ stain according to the manufacture’s protocol (BD Bioscience).
Assays to measure activity of pyruvate kinase, hexokinase, glucose-6-phosphate dehydrogenase, glucose phosphate isomerase, triose phosphate isomerase, glutathione peroxidase as well as concentration of reduced glutathione were performed as described by Beutler
Packed RBC were thawed on ice by addition of 3 volumes buffer (10 mM TRIS, pH 6.5; 2 mM EDTA; 10 mM dithiothreitol (DTT)) containing proteasome inhibitors (Complete ULTRA, Roche) for hypotonic lysis. After centrifugation, TRIS (pH6.5) was added to a final concentration of 100mM followed by passage over DEAE sephadex A-50 (GE Healthcare) to remove hemoglobin. Following three washes with 100 mM TRIS, pH 6.5, cytosolic proteins were removed from the column with 100 mM TRIS, pH 6.5, 0.5 M NaCl. The resulting protein solution was concentrated and subjected to buffer exchange with 10 mM TRIS pH6.5, 2 mM EDTA using centrifugal filter units (Millipore Ultracel 10k). Protein concentration was measure using BCA™ Protein Assay (Thermo Scientific) following the manufacturer’s protocol.
400 µg of cytosolic proteins were prepared for 2D electrophoresis using the Ready-Prep 2-D clean up Kit (Bio-Rad Laboratories), proteins were resuspended in 7 M urea (Sigma), 2 M thiourea (Sigma) 2% m/v ASB-14 (G-Bioscience) and 30 mM TRIS; protein concentration was assessed using 2-D Quant Kit (GE Healthcare). 300 µg of each sample were labeled using the Amersham™ 5 nmol CyDye Fluors minimal Dye labeling Kit according to the manufacturer’s protocol (GE Healthcare). We used a dye reversal labeling strategy: each sample was run twice in any given experiment labeled with Cy-3 and Cy-5, respectively. When possible the patient was paired with either parent to run on a gel for direct comparison. An equal amount of each sample in any given experiment was also pooled and labeled with Cy-2 to serve as an internal standard used to normalize expression levels. This also allowed us to compare between gels of a sample set. A standard sample was created as a bridge between experiments by pooling 10 blood samples from healthy donors. This common standard sample was included within each patient-specific DIGE experiment.
Using passive rehydration loading, 300 µl (300 µg pooled sample) were applied to 17 cm 3–10 NL immobilized pH gradient strips (Bio-Rad Laboratories) after adding carrier ampholytes (IPG buffer 3–10 NL, GE Healthcare) and 2 mM DTT (Bio-Rad Laboratories). Labeled proteins were isoelectrically focused using the PROTEAN IEF Cell (Bio-Rad Laboratories). Focusing parameters were 50–250V linear gradient for 1 hour, 250–4000 V linear gradient for two hours, and then hold at 8000V and hold for an accumulation of 56000 V-hours. IEF was temperature and current limited at 25°C and 50 µAmp per strip
Focused IEF strips were reduced and carbamidomethylated for 10 minutes at each step using 10 mg/ml DTT and 25 mg/ml iodoacetamide solutions (Equilibration Solutions I & II, Bio-Rad Laboratories) formulated in a 6 M urea/30% (v/v) glycerol/2% SDS 50 mM TRIS buffer at pH 8.8. The IEF strips were rinsed briefly in SDS-PAGE electrophoresis buffer (25 mM TRIS, 192 mM glycine, 0.1% SDS), and then placed on 10–14% gradient polyacrylamide gels cast in low-fluorescence glass plates (Jule Technologies). We removed 1 cm from the basic end of the strips to accommodate the dimensions of the gel chambers. Each IEF strip was fixed to the gel with warm 0.5% (w/v) agarose sealing solution (Bio-Rad Laboratories). PAGE was run overnight in a PROTEAN IIxi Cell at 14°C at 8 mA/gel for the first hour and 12 mA/gel for 15 hours. Power was continuously supplied until the bromophenol blue front migrated out the end of each gel.
Gels were documented with a Typhoon Trio imager (GE Healthcare). Fluorescence images were analyzed with Progenesis SameSpots software (version 3.2, Nonlinear Dynamics), differentially expressed spots were identified and a pick list was created. Spots with an average normalized volume (ANV)≤5000 or a spot area (SA) below 400 for HA09, SA≤250 for HA19 and SA≤300 for HA21 and HA24 were excluded from analysis based upon difficulty picking and identifying low abundance proteins. Between 50 - 69% of spots remaining had a variance between the sample groups (ANOVA)≤0.05 (see
Gel spot digests in 96-well plates were dried, and resuspended 50 µl/well using 5% (v/v) acetonitrile, 0.1% (v/v) formic acid (Buffer A). An Agilent dual pump system with a 2/6 switching valve was used to inject and elute the tryptic peptide solutions to a Thermo Finnegan LCQ Deca XP MAX ion trap mass spectrometer for protein identification. Agilent pump system components are an 1100 series Quaternary Pump, an 1100 series micro well plate sampler, a 2-position 6-port switching valve, and a 1200 series NanoFlow binary pump. During the sampling phase, 40 µl aliquots were injected to a 5 mm C18 trap column loop on the switching valve. The trapped peptides were then eluted with the NanoFlow pump at 0.5 µl/min to a custom-built in-lab 7 cm C18 (5 µm particle size, Jupiter C18, Phenomenex Corporation) reversed-phase 100 µm I.D. micro column. Peptides were resolved with linear gradient elution using 95% (v/v) acetonitrile, 0.1% (v/v) formic acid (Buffer B) as the organic phase: from 0% to 40% Buffer B over 30 minutes
Xcalibur 2.0 SR2 generated RAW files were extracted for MS2 information using RAWXtract version 1.9.9.1
Peptide identification was validated first by matching location on the gel (pH and molecular weight) with the theoretical isoelectric point (pI) and mass of the identified protein. Superimposing gel images (based on Coomassie stain) from all experiments showed that some spots had been picked in separate experiments, resulting in consistent protein identification(s). Spots with multiple IDs were dismissed when no clear predominant protein could be determined; when spectral counts of peptides from one protein exceeded 60% of total spectral counts in a spot, it was considered to be the predominant protein. Fold change was calculated by comparing measured expression level (normalized volume) of patient to the average of all other samples run in a set. For protein identification, we focused upon spots that showed≥1.5 fold difference (in either direction) between the patient and all other samples run in the experiment combined (“P to all”).
Cytosolic proteins were separated on 11% polyacrylamide gels and electro blotted onto nitrocellulose (BioRad, CA). Membranes were blocked in Odyssey® blocking buffer (LI-COR Biosciences). Primary antibodies used were as follows: rabbit polyclonal anti-actin (Sigma, MO), mAb anti PKLR (Santa Cruz Biotechnology, CA). Secondary antibodies used were IRDye® 800 goat anti-mouse and IRDye® 680 goat anti-rabbit, followed by detection with Odyssey® infrared imaging system and analysis with the application software version 3.0.21 (LI-COR Biosciences). This software was also used to perform densitometry analysis to normalize PKLR signal to loading control (actin).
Western blot analysis of PK expression in sample set HA24. Hemoglobin depleted RBC lysates from HA24 patient (P), mother (M), father (F), sister (S) and control sample (C) were analyzed for PK expression. Densitometry was calculated as percentage (%) of signal compared to the average of sister and control samples after normalizing to loading control actin.
(TIF)
Prototypical image of a Coomassie stained polyacrylamide gel includes protein localization markers for protein mass in kilodalton (Marker/kD) and isoelectric point (pI). All 243 picked spots were superimposed to create this image; spots picked in HA09 are shown in green, HA19 in purple, HA21 in blue and HA24 in red. Multicolored circles indicate spots that were picked in more than one experiment. Numbers refer to spot ranks in Supplementary Tables 3 and 4 (HA09, green), Supplementary Tables 5 and 6 (HA19, purple), Supplementary Tables 7 and 8 (HA21, blue) and Supplementary Tables 9 and 10 (HA24, red).
(TIF)
Exportin 7 protein expression in HA19, HA21 and HA24. Included are all spots in which Exportin-7 was identified as the predominant protein. A: Graph shows expression levels in normalized volume for both replicates run per indicated sample. B: Images show sections of the gels where Exportin 7 containing spots were excised (blue lines enclose spot areas; upper panel: HA19, middle panel: HA21, lower panel HA24). Pictures were exported from Same Spots and merged to show all relevant spots in one picture.
(TIF)
Fumarate Hydratase expression in HA19 and HA21. Included are all spots in which Fumarate Hydratase (FH) was identified as the predominant protein. A: Graph shows expression levels in normalized volume for both replicates run per sample. B: Images show sections of the gels where FH containing spots were excised (blue lines enclose spot areas; upper panel: HA19, middle panel: HA21). Pictures were exported from Same Spots and merged to show all relevant spots in one image per sample.
(TIF)
Complete Summary of Enzyme Assays performed on samples included in this study.
(DOCX)
Complete list of all Proteins identified.
(DOCX)
Proteins not listed in Red Blood Cell protein databases.
(DOCX)
A: Differentially expressed spots with expression level in Patient sample higher compared to controls. B: Differentially expressed spots with expression level in Patient sample lower compared to controls.
(DOCX)
Expression of Cytoskeleton Proteins.
(DOCX)
Expression levels of Chaperones.
(DOCX)
Expression of proteins involved in Protein Degradation/Proteasome/Ubiquitin pathway.
(DOCX)
Complete list of all spots picked and proteins identified in HA09.
(DOCX)
Complete list of all spots picked in HA19, fold change comparing patient to all other samples run in the experiment.
(DOCX)
Complete list of all spots picked and Proteins identified in HA19.
(DOCX)
Complete list of all spots picked in HA19, fold change comparing patient to all other samples run in the experiment.
(DOCX)
Complete list of all spots picked and Proteins identified in HA21.
(DOCX)
Complete list of all spots picked in HA21, fold change comparing patient to all other samples run in the experiment.
(DOCX)
Complete list of all spots picked and Proteins identified in HA24.
(DOCX)
Complete list of all spots picked in HA24, fold change is comparing patient to all other samples run in the experiment.
(DOCX)
We would like to thank Jeanine Witkowski and Deborah Noack for technical assistance. We thank Drs. Martin Campbell, Shipra Kaicker, Michael Armstrong and Ms. Donna Gierek and study participants for providing samples. We thank Dr. Kelly Bethel for review of peripheral blood smears.