Assessment of the Red Cell Proteome of Young Patients
with Unexplained Hemolytic Anemia by Two-
Dimensional Differential In-Gel Electrophoresis (DIGE)
Katharina von Lo ¨hneysen, Thomas M. Scott, Katrin Soldau, Xiuling Xu, Jeffrey S. Friedman*
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America
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.
Citation: von Lo ¨hneysen K, Scott TM, Soldau K, Xu X, Friedman JS (2012) Assessment of the Red Cell Proteome of Young Patients with Unexplained Hemolytic
Anemia by Two-Dimensional Differential In-Gel Electrophoresis (DIGE). PLoS ONE 7(4): e34237. doi:10.1371/journal.pone.0034237
Editor: Andy T. Y. Lau, Shantou University Medical College, China
Received October 25, 2011; Accepted February 24, 2012; Published April 3, 2012
Copyright: ? 2012 von Lo ¨hneysen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grant 1RO1 DK080232 awarded to JSF. Center for AIDS Research Support 3 P30 AI036214-13S1, is gratefully
acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
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 erythropoi-
etin 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 . Reticulocytes circulate for a few days during
which organelles including mitochondria, Golgi apparatus and the
endoplasmic reticulum are lost , allowing mature RBC maximal
flexibility to squeeze though narrow capillaries and providing
room to pack the cell with hemoglobin, ultimately making up 90%
of the dry weight of the cell . Because of their extreme
specialization, mature RBC have little capacity to repair and no
ability to replace damaged proteins. RBC are routinely exposed to
high oxygen concentrations and are vulnerable to the accumula-
tion of damage caused by oxidative stress - with much of their
metabolic activity devoted to reducing oxidative damage.
Therefore a highly reducing milieu and a stable redox balance
are paramount for proper function and cell survival. Major
components of RBC defense against reactive oxygen species
(ROS) include reduced glutathione (GSH) and enzymatic
antioxidants such as catalase, peroxiredoxins and superoxide
dismutase. Glycolysis and the pentose phosphate pathway are the
only sources for NADH and NADPH (respectively) needed to
protect RBC from oxidative damage. NADH is required for
reduction of methemoglobin; NADPH is utilized primarily for the
reduction of oxidized glutathione (GSSG ) 2GSH). Disruptions
in redox balance and increased oxidative damage are character-
istic of many RBC pathologies including hemoglobinopathies such
as sickle cell anemia  and thalassemia , as well as enzyme
defects such as glucose-6-phosphate dehydrogenase (G6PD)
deficiency  and pyruvate kinase (PK) deficiency .
Hereditary non-spherocytic hemolytic anemias (HNSHA) are a
heterogeneous group of RBC enzymatic disorders with PK and
G6PD deficiencies being the most common lesions . While
G6PD deficiency is the most common enzyme deficiency in
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humans, clinical phenotypes are quite variable depending upon
the severity of the underlying mutation, and are related to residual
enzymatic activity . Specific diagnosis of HNSHA is ap-
proached through testing activity of enzymes involved in glycolysis
and other RBC metabolic pathways. Unfortunately, no enzymatic
abnormality is found in up to 70% of cases, highlighting the need
for new approaches to understanding the etiology of this disorder
[10,11]. In order to better characterize HNSHA of unknown
etiology we have focused upon proteomic analysis of RBC. We
have employed a 2D-DIGE (2-dimensional difference gel
electrophoresis) approach to identify changes in expression
patterns of RBC cytosolic proteins from anemic patients when
compared with those of healthy relatives (parents and siblings) and
unaffected controls. The application of a comprehensive, unbiased
method to asses relative protein abundance broadens the spectrum
of knowledge of the RBC proteome and identifies candidate genes,
proteins and pathways that may play a role in abnormal RBC
Figure 1 illustrates the experimental scheme for the 2D-DIGE
approach used to identify proteins differentially expressed in RBC
cytosols. Routine evaluation of samples began with non-proteomic
approaches including testing for unstable hemoglobin and
evaluation of enzymatic activity for the most common causes of
HNSHA (G6PD, PK, and Hexokinase deficiency), if not
previously reported in the patient’s record. RBC preparation
included passage of cells over microcrystalline cellulose to remove
leukocytes followed by hypotonic lysis of RBC, removal of
membranes and hemoglobin depletion from the resulting
hemolysate. The resulting cytosolic protein fraction was then
prepared for DIGE. Gel images were evaluated, and differentially
expressed ‘spots’ were excised and processed for protein
identification by LC/MS.
Table 1 summarizes relevant laboratory evaluation of the 4
HNSHA patients included in this study: HA09, HA19, HA21 and
HA24 (HA for hemolytic anemia). HA09 is a 10-year old male
who presented with severe chronic hemolytic anemia at birth, and
who remains transfusion dependent. Bone marrow aspirate
revealed erythroid hyperplasia with prominent basophilic stip-
pling. Prior workup was negative for hemoglobinopathy, common
enzyme defects or RBC membrane abnormalities. Lab values
concurrent with sampling for this study included hematocrit
(27%), hemoglobin 8.9 g/dl, RBC count 3.09x1012/l, reticulocyte
count of 3.5%, bilirubin (11.0 mg/dl) and ferritin (639 ng/ml)
levels. Because of the patient’s transfusion dependence, the sample
was obtained at the nadir just prior to transfusion (in this case
6 weeks following the most recent transfusion) to maximize the
fraction of patient derived RBC for analysis. Glucose-6-phosphate
dehydrogenase (G6PD), pyruvate kinase (PK), hexokinase (HK),
gluthathione peroxidase (GPx), glutamate-oxaloacetate transami-
nase (GOT) and glucose phosphate isomerase (GPI) activity as well
as a screening test for pyrimidine 5’ nucleotidase (P5’N-1) were
evaluated to rule out known enzyme defects. Reduced Glutathione
(GSH) level was normal. Lacking a clear diagnosis, this sample was
selected for proteomic analysis comparing HA09 (patient), his
mother, one unrelated control, and a common pooled control
sample. The same, pooled control sample (designated ‘standard’)
was used in each subsequent HA DIGE experiment as a common
control for comparison between experiments.
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 (Table S1). Because no
diagnosis for the etiology of anemia was made, this sample was
selected for proteomic analysis comparing HA19 (patient), both
parents and one unrelated control sample.
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
Figure 1. Schematic illustrating the experimental work flow
from diagnosis of an anemia patient as HNSHA of unknown
etiology to identifications of proteins potentially involved in
the pathology of the disease.
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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 deficien-
cies, 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 (Table S1). Despite this ambiguity,
proteomic analysis was begun in this case comparing HA24
(patient), mother, father, sister and one unrelated control.
Subsequent sequence analysis of the PKLR gene revealed that
HA24 is homozygous for the R479H mutation ; the parents
are heterozygous carriers, while the sister is unaffected. A
subsequent western blot analysis utilizing a PK specific antibody
demonstrated low, but detectable PK expression in the patient,
and intermediate expression in both parents compared to
unaffected controls (Figure S1). Proteomic analysis of these
samples was completed, providing a comparison of differential
protein expression in the context of a defined molecular cause of
Table 2 provides an overview of DIGE experiments, including a
summary of the number of spots detected on gels from each
sample set after spot filtering, and the number of filtered spots
differentially expressed by analysis of variance (ANOVA). We
excluded spots from further evaluation when they were not
matched correctly between the gels, when spot intensity appeared
too low to allow identification by mass spec, and when a spot
contours drawn by software corresponded to a smear rather than a
spot with defined boundaries. A total of 243 spots were picked and
a total of 213 proteins were identified (several proteins being
repeatedly identified) when pooling data from all four experiments
(Table S2). 21 of these proteins have not been previously identified
in RBC proteome databases [13,14,15] (listed in Table S3).
Despite good resolution on 2-D gels, proteins with similar
molecular weight and isoelectric point (pI) comigrate, resulting
in identification of more than one protein in some spots analyzed.
As an overall data quality metric, Principal Component Analysis
(PCA, Figure 2) of all spots included (Table 2) shows that replicate
samples (run with dye reversal) are highly similar in all cases, and
that protein expression patterns in HA patient samples are easily
distinguished from control samples run in the same experiment.
Replicate samples remained tightly grouped when PCA was
performed on all detected spots or on all differentially expressed
spots (ANOVA). Plotting of the spots showed that in all cases the
patient was an ‘‘outlier’’, separated from more closely grouped
family members and controls.
Table 1. Sample characterization (Blood Parameters and
Normal RangeHA09**HA19 HA21 HA24**
Age (years)9.752 16.250.5
Red cell count
0 = negative
1 = positive
ENZYME ACTIVITY (UI/g HB)
Pyruvate Kinase 11.1– 18.913 220.127.116.11
G6PD 7.9 – 16.3 9.825.914.9ND
Hexokinase 1.02– 2.54 1.62 5.171.9 3.45
GPI38.8– 82.2 4986.360.6ND
TPI 1317– 2905 ND 1602 1387ND
21.3– 40.330.2ND 44 ND
4.5 – 18.104.22.168ND8.19
**blood transfusion prior to drawing blood: HA09 42 days, HA24 37 days.
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 Table 1.
Table 2. Summary of spots detected by Same Spots
Spots HA09HA19 HA21HA24
488 411687 580
Excluded 291256 477 471
Included197 155 210 109
5363 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
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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 (Figure 3, A). For HA19 and HA21 we found a
comparable number of differentially expressed protein spots with
elevated or reduced expression in the patient versus controls
(Figure 3, B-C). 25 spots showed reduced expression in HA24
compared to control samples in this set (Figure 3, D, lower panel).
We found 9 spots with increased expression in the patient
accompanied by intermediate expression levels in the mother
(Figure 3, D, upper panel). In each DIGE comparison, we
observed several protein spots with expression intensities for one or
both parents intermediate between patient and control samples (as
in Figure 3, D above), a pattern that might be expected if parents
are heterozygous for a polymorphism affecting expression of the
respective protein. Spots with this pattern are considered in more
detail below (see Table 3).
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 Figure S2.
Figure 4 presents an overview of this data highlighting proteins
that were most highly or most frequently differentially expressed.
These include exportin 7 (XPO7), fumarate hydratase (FH),
purine nucleoside phosphorylase (PNP), chaperones, cytoskeletal
and ribosomal proteins, proteasome subunits and additional
proteins involved in protein degradation. Proteins showing lower
expression in individual patients are listed in Supplementary Table
XPO7 is a member of the importin-beta superfamily of nuclear
transport receptors thought to cycle between the cytoplasm and
the nucleus . XPO7 was identified in multiple co-migrating
spots in three experiments (Figure 5, A and Figure S3) presumably
representing isoforms with distinct post-translational modifica-
tions. Patients HA19, HA21 and HA24, all showed consistently
increased levels of XPO7 relative to control samples.
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 (Figure 5, B and Figure S4) despite these patients
having the highest reticulocyte counts among samples examined.
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 [17,18]. Patients HA19 and HA21
both show lower PNP abundance in several spots when compared
to control samples. Interestingly, the extent of decreased
expression varies among the spots: in HA19 from 3.1 fold to 7.7
fold and in HA21 from 2.23, to 9.1 fold, respectively (Table S4, B),
while the overall expression pattern remains similar (Figure 5, C,
Abundance of specific cytoskeletal proteins differed in all four
patients (Supplementary Table S5–and Figure 4) compared to
controls. Actin was increased in patients HA09 and HA24, while
tubulin was increased in HA21, and a subunit of the dynactin/
dynein complex (ACTR1A)  showed higher levels of
expression HA09. Expression of an actin filament capping protein
(CAPZB), a regulator of actin filament growth  was found to
be lower in HA19. A small GTPase, RhoA, a regulator of actin
dynamics  showed lower expression in HA19 and HA21. We
also found diminished expression levels in these patients for two
GDP dissociation inhibitors that influence the GDP-GTP
exchange of rab GTPases, known to be involved in vesicle
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  and cancer cells
[24,25]. In HA09 PA2G4 expression is reduced 1.5 to 1.61 fold
(Table S4, B). Ribosomal protein SA (RPSA), also called Laminin-
Figure 2. Principal Component Analysis was calculated by
Same Spots software. Dot plot of all spots included in analysis (see
Table 2) shows that in all four experimental sets (HA09, HA19, HA21 and
HA24), replicate samples group closely together and patient samples
differ significantly from both familial and unrelated control samples.
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receptor 1, was more highly expressed in HA19, HA21 and HA24
(Table S4, A). In addition to being a receptor for laminin, RPSA is
required for assembly and stability of the 40S ribosomal subunit.
Chaperones were also differentially expressed in all patients
(Table S6 and Figure 4). Conspicuously, in HA09 multiple T-
complex subunits (TCP) showed reduced expression, while several
heat shock proteins (HSP) were present in higher amounts when
compared with control samples (Figure 6, A). In HA19 and HA21
several TCP subunits were increased; HA19 also had decreased
expression of a single heat shock related protein (HSPA8, Figure 6,
B). Several chaperone proteins showed reduced expression in
HA19 and HA24, although only two spots in HA21 and one spot
in HA24 met the threshold for a change above 1.5-fold.
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 (Figure 7, Table S7), indicating differences in protein
turnover between HA patient RBC and control samples. There
was a single exception to this pattern among the proteasome
subunits (increased expression of PSMC5 in patient HA21,
Figure 7, C green line). In HA19 and HA21 two additional
proteins associated with the proteasome, thioredoxin-like 1 
and PITHD1 (C-terminal proteasome-interacting domain of
thioredoxin-like) domain-containing protein 1, showed higher
expression levels compared to controls.
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 (Table 3 and Figure 8)–particularly in
those cases where expression was decreased in the HA patient
sample. Such patterns of possible recessive inheritance were
observed in patients HA09, HA21, and HA24.
In HA09 (Figure 8, A) two spots containing G6PD (#82 and
#160) and two spots identified as PA2G4 (#232 and #307), were
identified with the lowest expression measured in the patient
sample, with mother or mother and father showing intermediate
expression relative to unrelated controls. Expression profiles for
HA19 are shown in Figure 8, B. 14-3-3 epsilon (#94) showed
reduced expression in both the patient and mother, and HSPA8
(#89) showed low expression in the patient with intermediate
expression in both parents. PITHD1 (#231) showed increased
expression in the patient, and intermediate expression in the
mother. In HA21, proteasome subunit PSMB4 (#61) and
superoxide dismutase 1 (#204) showed low expression in the
patient and intermediate expression in the mother (Figure 8, C,
left panel). Four spots with higher expression in the patient and
intermediate expression in the mother compared to controls
(Figure 8, C, right panel) were identified: EIF2S3 (eukaryotic
translation initiation factor, #28), ACTR1A (alpha-centractin,
#67), and two spots both identified as VCP (valosin containing
protein, #162 and #200).
In HA24, CCT8 (T-complex chaperone subunit, #97) was
identified as a protein with low expression in both the patient and
mother (Figure 8, D, left panel). 8 spots with increased expression
in the patient and intermediate expression in one parent were
identified: RPSA (ribosomal protein SA, #13), XPO7 (exportin 7,
#14 and #55), EEF2 (elongation factor 2, #4) BPGM (bispho-
sphoglycerate mutase, #36 and #43), ADSL (adenylosuccinate
Figure 3. Expression profiles of spots showing up- or down-regulation in patient samples. Of all spots included in the analyses (see
Table 2), those showing a distinct up-regulation (P , upper panels) or down-regulation (P , lower panels) in the patient sample when compared with
all other samples of the same experimental sets are shown in A: HA09, B: HA19, C: HA21 and D: HA24. Graphic depictions of expression levels include
both replicates run per sample comparing normalized volumes.
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lyase, #132), PRDX2 (peroxiredoxin 2, #159) and PGAM1
(phosphoglycerate mutase 1, #167).
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 [11,27]. However, arriving at a molecular
diagnosis in patients with HNSHA remains a challenge, with the
majority of clinical evaluations failing to identify a causal lesion–
indicating that novel approaches to diagnosis are required. In this
study, we tested whether a quantitative proteomic approach
utilizing 2-D gels could be used to discriminate between anemic
and control RBC proteomes in four patients with HNSHA, and
provide identification of specific proteins and pathways that may
be involved in pathogenesis in individual patients. We found
several proteins that might serve as biomarkers for HNSHA,
however, our findings are preliminary and further validation is
needed in order to establish a clear link between those proteins
and the etiology of anemia.
Each sample was evaluated on 2 separate gels using a dye
reversal approach [28,29] to avoid systematic bias in labeling or
detection of protein spots, and to allow each patient sample to be
compared against parental samples (when available) within the
same gel. Principal component analysis showed a close grouping of
all replicate samples with patient samples clearly distinct from
controls and healthy family members (Figure 2). Several spots were
picked independently as differentially expressed in more than one
patient/family comparison. In all such instances, the same
protein(s) was identified, showing that our peptide identification
was reliable and reproducible (Figure S2 and corresponding
Tables S8, S9, S10, S11, S12, S13, S14, S15). Low abundance
spots were not chosen for picking because we could not reliably
identify them based upon the sensitivity of our mass spec. In total
we identified 213 proteins in 243 spots picked for analysis (Table 2),
Table 3. Proteins with parental expression intermediate between patient and unrelated control samples.
ID Spot rank Fold changeGene Protein
82 - 2.63G6PD Glucose-6-phosphate 1-dehydrogenase
CAP1Adenylyl cyclase-associated protein 1
160 - 1.93G6PD Glucose-6-phosphate 1-dehydrogenase
232 - 1.73PA2G4 Proliferation-associated protein 2G4
307- 1.61PA2G4 Proliferation-associated protein 2G4
89 - 2.14HSPA8 Isoform 1 of Heat shock cognate 71kD
94 - 1.92 LOC44091 Similar to 14-3-3 protein epsilon
+1.68PITHD1 PITH domain-containing protein 1
EIF2S3Eukaryotic translation initiation factor 2, subunit 3
61PSMB4Proteasome subunit beta type-4 (precursor)
204 -1.93SOD1 Superoxide dismutase 1, soluble
+1.89HDHD2 Haloacid dehalogenase-like hydrolase domain containing 2
EEF2 Elongation factor 2
13RPSA Ribosomal protein SA
14XPO7 Exportin 7
36BPGM Bisphosphoglycerate mutase
43 BPGM Bisphosphoglycerate mutase
55XPO7 Exportin 7
97-1.76 CCT8 Chaperonin containing TCP1, subunit 8
ADSL Isoform 1 of Adenylosuccinate lyase
159 PRDX2Peroxiredoxin 2
167PGAM1Phosphoglycerate 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.
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21 of which are not included in previously published RBC
proteome databases (Table S3) [13,14,15].
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 , may
simply be a reflection of reticulocytosis and not be directly related
to the underlying pathology causing hemolysis. While reticulocytes
were elevated in all HA patients examined relative to controls,
HA19 and HA21 had the highest reticulocyte counts at 11.6% and
18.4%, respectively–and these two samples had the highest
number of spots showing increased expression relative to controls
(Table S4, A). Included in the list of proteins with increased
expression in both patients were several involved in protein
synthesis, the heme biosynthetic enzyme uroporphyrinogen
decarboxylase (UROD), autophagy related 3 (ATG3) protein that
is an important regulator of mitochondrial homeostasis , and
several subunits of the TCP1 chaperone complex that aids in
folding actin and tubulin among other targets [32,33].
Exportin 7 (XPO7), a protein involved in shuttling macromol-
ecules across the nuclear envelope , was also increased in both
patient samples. Interestingly, XPO7 was recently identified as a
protein essential for proper erythroid maturation in mammals
. Deficiency of XPO7 could thus presumably lead to inferior
RBC integrity caused by a defective maturation process, and low
expression of this protein could play a role in the etiology of
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 , shows
increased expression in cancer cells where expression correlates
with invasiveness and metastasis .
Eukaryotic elongation factor 2 (eEF2), a protein that mediates
mRNA translation at ribosomes, was increased in patients HA21
and HA24 (Tables S12, S13, S14 and S15). In contrast, HA09, the
patient with the lowest reticulocyte count, showed decreased
expression of many of the same proteins when compared with
control samples (Table S4, B).
Patient HA09 is also transfusion dependent, and the combina-
tion 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.
Protein Quality Control
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 . The observation of differential expression of
proteins involved in endogenous protein quality control and
degradation pathways in HA patient samples may be related to
increased reticulocytosis (as discussed above), or may be a response
to an increased load of misfolded or damaged proteins that
accumulate in developing RBC of HA patients. Chaperones, like
heatshock proteins (HSP), can refold polypeptides and salvage
them; when proteins are irreparably damaged they ensure their
degradation by the ubiquitin-proteasome pathway . HSP play
a key role in the triage of proteins with oxidative damage [39,40],
are normally downregulated during erythroid maturation , but
show increased expression in the context of RBC defects such as
thalassemia . Interestingly, we identified multiple differentially
expressed protein species in all HA patient samples that are
components of both endogenous protein quality control and
protein degradation systems (Figure 4, Tables S6, S7).
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 (Table S7), and represents
perhaps the clearest evidence of phenotypic similarity between
samples. Among the 4 HA samples analyzed, there was a
divergence in the pattern of differential expression of HSP and
chaperone proteins–most notably HA19 and HA21 showed
increased expression of T-complex chaperone proteins while
HA24 and in particular HA09 showed decreased expression of T-
complex proteins and increased expression of several HSP
(Table S6). The cytosolic TCP-1 ring complex (TRiC; also called
CCT, for chaperonin containing TCP-1) consists of 8 subunits
, assists in the folding of numerous proteins, and is often found
Figure 4. Schematic depiction summarizing proteins and
groups of proteins found to be differentially expressed in
patients HA09, HA19, HA21 and HA24. Proteins that were
expressed at higher levels in patients are represented by green ovals,
while down-regulated proteins are represented by red ovals.
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associated with nascent polypeptides at ribosomes . In
addition, we found increased expression of an additional protein
associated with the ubiquitin-proteasome- system that also exhibits
chaperone activity, valosin containing protein (VCP) , in
HA21 (Figure 7, C). Increased expression of VCP has been shown
to counteract the toxicity of both bortezomib on proteasome
activity and geldanomycin on HSP function . Higher
expression of HSPs and VCP may be a compensatory response
to decreased activity of the proteasome machinery; HSPs and
VCP may function together to enhance chaperone activity and
avoid accumulation of misfolded proteins. Compounds that
increase chaperone activity have potential as therapeutic agents
Figure 5. Exportin 7, Fumarate Hydratase and Purine Nucleoside Phosphorylase protein expression. 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.
Hemolytic Anemia Comparative Proteomics
PLoS ONE | www.plosone.org8 April 2012 | Volume 7 | Issue 4 | e34237
by increasing the fraction of properly folded target proteins during
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 ; tubulin and actin are degraded by the ubiquitin –
proteasome pathway and are absent from the cytosol of mature
RBC in mice . We found both cytoskeletal proteins to be
increased in HA patients compared to controls. We also observed
a lower abundance of RhoA and RhoC as well as Rab GDP
dissociation inhibitor 1 and 2 (GDI 1/2) in patients HA19 and
HA21. Rho GTPases control actin dynamics , Rab GTPases
coordinate vesicle transport  and membrane trafficking .
Diminished availability of regulatory GTPases can interfere with
membrane reorganization and vesicle trafficking and could alter
actin dynamics in the maturation process from reticulocyte to
mature RBC and thus give rise to RBC with shortened life span.
Fumarate hydratase (FH) is a tricarboxylic cycle (TCA) enzyme
with reduced expression in patients HA19 and HA21 (Figure 5, B,
Supplementary Figure S4) even though both patients had a high
reticulocyte count (Table 1). Because of its mitochondrial function
and localization, FH is not expected to be part of the mature
erythrocyte’s proteome, yet it was previously identified in RBC
proteome studies [13,14,15], suggesting an unknown function
outside of the TCA cycle. A relationship between heme synthesis,
Figure 6. Expression profiles of spots predominantly containing Chaperone proteins are shown (see also Supplementary Table S6).
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.
Figure 7. Expression profiles of proteins involved in protein degradation (see also Supplementary Table S7). 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).
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heme catabolism and FH activity has been identified in tumor cells
where loss of FH activity is synthetically lethal with defects in heme
catabolism . In this context, heme synthesis utilizes TCA
substrates proximal to FH, overcoming a TCA cycle defect
allowing production of required NADP and maintenance of
mitochondrial membrane potential. Heme catabolism provides a
sink allowing this bypass to continue. The function of FH in the
cytosol of mature RBC, and the functional impact of the decline in
FH expression found in HA19 and HA21 is unclear. One
possibility is that during RBC development, reduced FH activity
slows distal metabolite flow in the TCA cycle, thus making
succinyl-CoA available for heme biosynthesis. The observed
reduction in FH protein in peripheral RBC of two patients in
this study may thus be a downstream consequence of altered
development. This would indicate an even more pronounced
reduction in FH expression in marrow progenitors of these
Purine Nucleoside Phosphorylase
HA19 and HA21 also showed reduction in expression of purine
nucleoside phosphorylase (PNP, Figure 5, C and D), an enzyme
highly expressed in early erythroid precursors and erythrocytes,
that is an integral part of the purine salvage pathway (http://
biogps.org/#goto=genereport&id=4860). PNP deficiency is life-
threatening condition resulting in severe T-cell dysfunction
associated with hypouricemia [52,53,54] and anemia [17,55,56].
Erythrocytes from patients with PNP deficiency show a depletion
of GTP and disruption of nucleotide pools . While neither
HA19 nor HA21 show evidence of immune dysfunction
characteristic of PNP deficiency, the degree of reduction of PNP
expression in these patients is dramatic, and may be related to the
etiology of their hemolytic anemia.
Limitations of the DIGE approach
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 (Table S1). Sequencing confirmed that HA24
was homozygous and both parents heterozygous for the R479H
mutation, previously identified as a cause of severe HNSHA
[12,58]. The mutation, located at the end of exon 10, appears to
interfere with mRNA splicing, decreasing protein production,
resulting in PK deficiency . Low PK activity in HA24 was
corroborated by low protein on Western-blot analysis (Figure S1).
However, we did not independently identify PK as a differentially
expressed protein using the DIGE approach, providing a clear
example of the limitations of this methodology.
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 compari-
Figure 8. Examples of protein expression patterns where one or both parents are intermediate between the levels measured in the
patient sample and those measured in the controls (see also Table 3). 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 Table 3.
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PLoS ONE | www.plosone.org10 April 2012 | Volume 7 | Issue 4 | e34237
sons, 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 (Figure S3), FH (Figure S4) and PNP
(Figure 5, D). While changes in molecular mass were minor, the
isoelectric points were different enough to create distinct spots.
Modifications such as phosphorylation, acetylation, oxidation and
proteolytic cleavage are presumed to underlie the observed
differences in isoelectric point . Shifting between different
isoforms creates a situation where overall expression of a protein
might not be changed, but volume measured could shift between
spots. Finally, altered protein expression does not necessarily
translate to changes in measured enzymatic activity. Bispho-
sphoglycerate mutase (BPGM) is an example for this disconnect
between measured expression levels and enzyme activity. BPGM
catalyses the synthesis of 2,3-disphosphoglycerate (2,3-DPG), that
binds to and regulates hemoglobin oxygen affinity. We found
BPGM protein levels were increased in samples from patient
HA24 and her mother (Table 3, Figure 8). However, when
assaying hemolysates derived from these samples, we found no
corresponding changes in BPGM enzymatic activity (data not
Evaluation and treatment of patients with hereditary nonspher-
ocytic 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
(Table S4, A, B). While DIGE is a powerful tool to identify
differential protein expression, drawbacks of this approach include
labor and resource intensity, as well as non-comprehensive nature
of the data obtained–in this case failure to identify PK as a
differentially expressed protein in patient HA 24. As such, this
approach is better suited to identifying qualitative differences
between samples, such as altered expression of proteins involved in
protein homeostatic systems, rather than as a specific diagnostic
tool for individual patients.
Materials and Methods
Sample Collection and Ethics Statement
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.
Packed Red Blood Cells
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 -80uC prior to fractionation
for proteomic analysis. Reticulocyte count was assessed using BD
Retic-CountTMstain according to the manufacture’s protocol (BD
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 . Unstable hemoglobin was determined by as
described by Carrell and Kay . A screening test for pyrimidine
5’ nucleotidase deficiency consisting of measurement of the
OD260/280 ratio of a perchloric acid extract of RBC was
performed as described by Beutler  on sample HA09 due to
the presence of basophilic stippling on peripheral smear. The ratio
was within the normal range.
Preparation of Cytosolic Proteins
Packed RBC were thawed on ice by addition of 3 volumes
buffer (10 mM TRIS, pH 6.5; 2 mM EDTA; 10 mM dithiothre-
itol (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 BCATM
Protein Assay (Thermo Scientific) following the manufacturer’s
Protein Preparation and Labeling for DIGE
400 mg of cytosolic proteins were prepared for 2D electropho-
resis using the Ready-Prep 2-D clean up Kit (Bio-Rad Labora-
tories), 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 mg of each sample were labeled using the
AmershamTM5 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
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Iso-Electric Focusing (IEF)
Using passive rehydration loading, 300 ml (300 mg 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 25uC and 50 mAmp per strip
Second-Dimension Polyacrylamide Gel Electrophoresis
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 14uC 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), differen-
tially 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 Table 2). Spots were also excluded
from analysis when they were not exactly matched between gels or
were too close to the edge of the gels (and thus not reliably
focused). Spots were sorted by ascending intensity to reduce carry
over from robotic gel excision. Gels were subsequently fixed in
10% methanol, 7% acetic acid (Fisher Scientific) and stained with
BioSafe Coomassie (Bio-Rad). Selected spots were picked with a
ProPicII picking robot (Genomic Solutions), followed by a
sequencing grade trypsin digest (Promega) in a ProGest digestion
station (Genomic Solutions).
Identification of Peptides by Liquid Chromatography -
Mass Spectrometry (LC-MS)
Gel spot digests in 96-well plates were dried, and resuspended
50 ml/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 ml 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 ml/min to a custom-built in-lab 7 cm
C18 (5 mm particle size, Jupiter C18, Phenomenex Corporation)
reversed-phase 100 mm 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 . To minimize sample carry-over,
the trap and column were cleaned by a 5-minute wash of Buffer B
after every injection. Tandem mass spectrometry was used to
characterize the peptides. The three highest abundance MS1
peaks were selected for CID fragmentation and MS2 for
Bioinformatic Processing and Analysis of MS Data
Xcalibur 2.0 SR2 generated RAW files were extracted for MS2
information using RAWXtract version 22.214.171.124 . MS2 files
were searched using the SEQUEST algorithm . SEQUEST
searches were performed using a concatenated target/decoy non-
redundant variant of the human IPI database version 3.33
(available for download at http://www.scripps.edu/chemphys/
cravatt/protomap) allowing for differential methionine oxidation
(+16 amu) and requiring cysteines to be carbamidomethylated
(+57 amu) . SEQUEST data from each gel spot were filtered
and sorted with DTASelect2 . Using DTASelect2 spectral
counting functions, identifications were checked for protein co-
location in gel spots. Additionally, sample carry-over effects
(generally due to contamination at the gel picker excision head)
could be monitored.
Evaluation of Data
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’’).
Western Blot Analysis
Cytosolic proteins were separated on 11% polyacrylamide gels
and electro blotted onto nitrocellulose (BioRad, CA). Membranes
were blocked in OdysseyH 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 IRDyeH 800 goat anti-
mouse and IRDyeH 680 goat anti-rabbit, followed by detection
with OdysseyH 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).
HA24. Hemoglobin depleted RBC lysates from HA24 patient (P),
Western blot analysis of PK expression in sample set
Hemolytic Anemia Comparative Proteomics
PLoS ONE | www.plosone.org12 April 2012 | Volume 7 | Issue 4 | e34237
signal compared to the average of sister and control samples after
normalizing to loading control actin.
amide 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).
Prototypical image of a Coomassie stained polyacryl-
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.
Exportin 7 protein expression in HA19, HA21 and
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.
Fumarate Hydratase expression in HA19 and HA21.
samples included in this study.
Complete Summary of Enzyme Assays performed on
Complete list of all Proteins identified.
Proteins not listed in Red Blood Cell protein
in Patient sample higher compared to controls. B: Differentially
expressed spots with expression level in Patient sample lower
compared to controls.
A: Differentially expressed spots with expression level
Expression of Cytoskeleton Proteins.
Expression levels of Chaperones.
Expression of proteins involved in Protein Degrada-
Complete list of all spots picked and proteins identified
comparing patient to all other samples run in the experiment.
Complete list of all spots picked in HA19, fold change
identified in HA19.
Complete list of all spots picked and Proteins
change comparing patient to all other samples run in the
Complete list of all spots picked in HA19, fold
identified in HA21.
Complete list of all spots picked and Proteins
change comparing patient to all other samples run in the
Complete list of all spots picked in HA21, fold
identified in HA24.
Complete list of all spots picked and Proteins
change is comparing patient to all other samples run in the
Complete list of all spots picked in HA24, fold
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
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.
1. Hoffbrand AV, Moss PAH, Pettit JE (2011) Essential haematology. Malden,
Mass.: Wiley-Blackwell. xi, 454.
Koury MJ, Koury ST, Kopsombut P, Bondurant MC (2005) In vitro maturation
of nascent reticulocytes to erythrocytes. Blood 105: 2168–2174.
Sivilotti ML (2004) Oxidant stress and haemolysis of the human erythrocyte.
Toxicol Rev 23: 169–188.
Banerjee T, Kuypers FA (2004) Reactive oxygen species and phosphatidylserine
externalization in murine sickle red cells. Br J Haematol 124: 391–402.
Origa R, Galanello R (2011) Pathophysiology of beta thalassaemia. Pediatr
Endocrinol Rev 8 Suppl 2: 263–270.
Fibach E, Rachmilewitz E (2008) The role of oxidative stress in hemolytic
anemia. Curr Mol Med 8: 609–619.
7.David O, Sacchetti L, Vota MG, Comino L, Perugini L, et al. (1990) Pyrimidine
5’-nucleotidase and oxidative damage in red blood cells transfused to beta-
thalassemic children. Haematologica 75: 313–318.
Baronciani L, Bianchi P, Zanella A (1996) Hematologically important
mutations: red cell pyruvate kinase (1st update). Blood Cells Mol Dis 22:
Prchal JT, Pastore YD (2004) Erythropoietin and erythropoiesis: polycythemias
due to disruption of oxygen homeostasis. Hematol J 5 Suppl 3: S110–113.
10. Beutler E, Lichtman MA, Beutler E, Kipps TJ, Seligsohn U, et al. (2006)
Disorders of red cells resulting from enzyme abnormalities. Williams
Hematology. New York: McGraw-Hill. pp 603–631.
Hemolytic Anemia Comparative Proteomics
PLoS ONE | www.plosone.org13 April 2012 | Volume 7 | Issue 4 | e34237
11. Miwa S, Fujii H (1996) Molecular basis of erythroenzymopathies associated with Download full-text
hereditary hemolytic anemia: tabulation of mutant enzymes. Am J Hematol 51:
12. Kanno H, Ballas SK, Miwa S, Fujii H, Bowman HS (1994) Molecular
abnormality of erythrocyte pyruvate kinase deficiency in the Amish. Blood 83:
13. Bouyssie D, Gonzalez de Peredo A, Mouton E, Albigot R, Roussel L, et al.
(2007) Mascot file parsing and quantification (MFPaQ), a new software to parse,
validate, and quantify proteomics data generated by ICAT and SILAC mass
spectrometric analyses: application to the proteomics study of membrane
proteins from primary human endothelial cells. Mol Cell Proteomics 6:
14. Goodman SR, Kurdia A, Ammann L, Kakhniashvili D, Daescu O (2007) The
human red blood cell proteome and interactome. Exp Biol Med (Maywood) 232:
15. Pasini EM, Kirkegaard M, Mortensen P, Lutz HU, Thomas AW, et al. (2006)
In-depth analysis of the membrane and cytosolic proteome of red blood cells.
Blood 108: 791–801.
16. Mingot JM, Bohnsack MT, Jakle U, Gorlich D (2004) Exportin 7 defines a novel
general nuclear export pathway. EMBO J 23: 3227–3236.
17. Markert ML (1991) Purine nucleoside phosphorylase deficiency. Immunodefic
Rev 3: 45–81.
18. Markert ML, Finkel BD, McLaughlin TM, Watson TJ, Collard HR, et al. (1997)
Mutations in purine nucleoside phosphorylase deficiency.Hum Mutat 9: 118–121.
19. Clarkson YL, Gillespie T, Perkins EM, Lyndon AR, Jackson M (2010) Beta-III
spectrin mutation L253P associated with spinocerebellar ataxia type 5 interferes
with binding to Arp1 and protein trafficking from the Golgi. Hum Mol Genet
20. Teumer A, Rawal R, Homuth G, Ernst F, Heier M, et al. (2011) Genome-wide
association study identifies four genetic loci associated with thyroid volume and
goiter risk. Am J Hum Genet 88: 664–673.
21. Kaibuchi K, Kuroda S, Amano M (1999) Regulation of the cytoskeleton and cell
adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem
22. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol
Cell Biol 10: 513–525.
23. Squatrito M, Mancino M, Donzelli M, Areces LB, Draetta GF (2004) EBP1 is a
nucleolar growth-regulating protein that is part of pre-ribosomal ribonucleo-
protein complexes. Oncogene 23: 4454–4465.
24. Zhang Y, Ali TZ, Zhou H, D’Souza DR, Lu Y, et al. (2010) ErbB3 binding
protein 1 represses metastasis-promoting gene anterior gradient protein 2 in
prostate cancer. Cancer Res 70: 240–248.
25. Hamburger AW (2008) The role of ErbB3 and its binding partners in breast
cancer progression and resistance to hormone and tyrosine kinase directed
therapies. J Mammary Gland Biol Neoplasia 13: 225–233.
26. Andersen KM, Madsen L, Prag S, Johnsen AH, Semple CA, et al. (2009)
Thioredoxin Txnl1/TRP32 is a redox-active cofactor of the 26 S proteasome.
J Biol Chem 284: 15246–15254.
27. Hirono A, Iyori H, Sekine I, Ueyama J, Chiba H, et al. (1996) Three cases of
hereditary nonspherocytic hemolytic anemia associated with red blood cell
glutathione deficiency. Blood 87: 2071–2074.
28. Karp NA, Kreil DP, Lilley KS (2004) Determining a significant change in
protein expression with DeCyder during a pair-wise comparison using two-
dimensional difference gel electrophoresis. Proteomics 4: 1421–1432.
29. Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in
protein expression: experimental design using minimal CyDyes. Proteomics 5:
30. Gilligan DM, Finney GL, Rynes E, Maccoss MJ, Lambert AJ, et al. (2011)
adducinknockoutmousemodelof hemolyticanemia.BloodCellsMol Dis47:85–94.
31. Radoshevich L, Debnath J (2011) ATG12-ATG3 and mitochondria. Autophagy
32. Chen X, Sullivan DS, Huffaker TC (1994) Two yeast genes with similarity to
TCP-1 are required for microtubule and actin function in vivo. Proc Natl Acad
Sci U S A 91: 9111–9115.
33. Yaffe MB, Farr GW, Miklos D, Horwich AL, Sternlicht ML, et al. (1992) TCP1
complex is a molecular chaperone in tubulin biogenesis. Nature 358: 245–248.
34. Hattangadi S, Burke K, Eng J, Cooney JD, Wang J, et al. (2011) Exportin 7
(RanBP16) Plays An Essential Role in Terminal Erythroid Chromatin
Condensation and Enucleation. ASH Annual Meeting Abstracts 118: 178-.
35. Malygin AA, Babaylova ES, Loktev VB, Karpova GG (2011) A region in the C-
terminal domain of ribosomal protein SA required for binding of SA to the
human 40S ribosomal subunit. Biochimie 93: 612–617.
36. NelsonJ,McFerranNV,PivatoG,ChambersE,DohertyC,etal.(2008)The67 kDa
laminin receptor: structure, function and role in disease. Biosci Rep 28: 33–48.
37. Sharma SK, Christen P, Goloubinoff P (2009) Disaggregating chaperones: an
unfolding story. Curr Protein Pept Sci 10: 432–446.
38. Khalil AA, Kabapy NF, Deraz SF, Smith C (2011) Heat shock proteins in
oncology: Diagnostic biomarkers or therapeutic targets? Biochim Biophys Acta
1816: 89–104. pp 89–104.
39. Pratt WB, Morishima Y, Peng HM, Osawa Y (2010) Proposal for a role of the
Hsp90/Hsp70-based chaperone machinery in making triage decisions when
proteins undergo oxidative and toxic damage. Exp Biol Med (Maywood) 235:
40. Ghosh N, Ghosh R, Mandal SC (2011) Antioxidant protection: A promising
therapeutic intervention in neurodegenerative disease. Free Radic Res 45:
41. Patterson ST, Li J, Kang JA, Wickrema A, Williams DB, et al. (2009) Loss of
specific chaperones involved in membrane glycoprotein biosynthesis during the
maturation of human erythroid progenitor cells. J Biol Chem 284:
42. Bhattacharya D, Saha S, Basu S, Chakravarty S, Chakravarty A, et al. (2010)
Differential regulation of redox proteins and chaperones in HbEbeta-
thalassemia erythrocyte proteome. Proteomics Clin Appl 4: 480–488.
43. Kubota H (2002) Function and regulation of cytosolic molecular chaperone
CCT. Vitam Horm 65: 313–331.
44. McCallum CD, Do H, Johnson AE, Frydman J (2000) The interaction of the
chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with
ribosome-bound nascent chains examined using photo-cross-linking. J Cell Biol
45. Wang Q, Song C, Li CC (2004) Molecular perspectives on p97-VCP: progress in
understanding its structure and diverse biological functions. J Struct Biol 146:
46. Mimnaugh EG, Xu W, Vos M, Yuan X, Neckers L (2006) Endoplasmic
reticulum vacuolization and valosin-containing protein relocalization result from
simultaneous hsp90 inhibition by geldanamycin and proteasome inhibition by
velcade. Mol Cancer Res 4: 667–681.
47. Chasis JA, Prenant M, Leung A, Mohandas N (1989) Membrane assembly and
remodeling during reticulocyte maturation. Blood 74: 1112–1120.
48. Liu J, Guo X, Mohandas N, Chasis JA, An X (2010) Membrane remodeling
during reticulocyte maturation. Blood 115: 2021–2027.
49. Ridley AJ (2006) Rho GTPases and actin dynamics in membrane protrusions
and vesicle trafficking. Trends Cell Biol 16: 522–529.
50. Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic
and cell physiology. Physiol Rev 91: 119–149.
51. Frezza C, Zheng L, Folger O, Rajagopalan KN, MacKenzie ED, et al. (2011)
Haem oxygenase is synthetically lethal with the tumour suppressor fumarate
hydratase. Nature 477: 225–228.
52. van Kuilenburg AB, Zoetekouw L, Meijer J, Kuijpers TW (2010) Identification
of purine nucleoside phosphorylase deficiency in dried blood spots by a non-
radiochemical assay using reversed-phase high-performance liquid chromatog-
raphy. Nucleosides Nucleotides Nucleic Acids 29: 466–470.
53. Hallett RJ, Cronin SM, Morgan G, Duley JA, Fairbanks LD, et al. (1994)
Normal uric acid concentrations in a purine nucleoside phosphorylase (PNP)
deficient child presenting with severe chicken pox, possible immunodeficiency
and developmental delay. Adv Exp Med Biol 370: 387–389.
54. Carpenter PA, Ziegler JB, Vowels MR (1996) Late diagnosis and correction of
purine nucleoside phosphorylase deficiency with allogeneic bone marrow
transplantation. Bone Marrow Transplant 17: 121–124.
55. Notarangelo LD (2009) Primary immunodeficiencies (PIDs) presenting with
cytopenias. Hematology Am Soc Hematol Educ Program. pp 139–143.
56. Giblett ER, Ammann AJ, Wara DW, Sandman R, Diamond LK (1975)
Nucleoside-phosphorylase deficiency in a child with severely defective T-cell
immunity and normal B-cell immunity. Lancet 1: 1010–1013.
57. Simmonds HA, Watson AR, Webster DR, Sahota A, Perrett D (1982) GTP
depletion and other erythrocyte abnormalities in inherited PNP deficiency.
Biochem Pharmacol 31: 941–946.
58. Kanno H, Wei DC, Chan LC, Mizoguchi H, Ando M, et al. (1994) Hereditary
hemolytic anemia caused by diverse point mutations of pyruvate kinase gene
found in Japan and Hong Kong. Blood 84: 3505–3509.
59. Valentini G, Chiarelli LR, Fortin R, Dolzan M, Galizzi A, et al. (2002) Structure
and function of human erythrocyte pyruvate kinase. Molecular basis of
nonspherocytic hemolytic anemia. J Biol Chem 277: 23807–23814.
60. Zhu K ZJ, Lubman DM, Miller FR, Barder TJ (2005) Protein pI shifts due to
posttranslational modifications in the separation and characterization of
proteins. Anal Chem 77: 2745–2755.
61. Beutler E (1984) Red Cell Metabolism: A Manual of Biochemical Methods. New
York, NY: Grune & Stratton, Inc.
62. Carrell RW, Kay R (1972) A simple method for the detection of unstable
haemoglobins. Br J Haematol 23: 615–619.
63. Friedman DB, Hoving S, Westermeier R (2009) Isoelectric focusing and two-
dimensional gel electrophoresis. Methods Enzymol 463: 515–540.
64. Lee IN, Chen CH, Sheu JC, Lee HS, Huang GT, et al. (2005) Identification of
human hepatocellular carcinoma-related biomarkers by two-dimensional
difference gel electrophoresis and mass spectrometry. J Proteome Res 4:
65. McDonald WH, Tabb DL, Sadygov RG, MacCoss MJ, Venable J, et al. (2004)
MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for
the storage of shotgun proteomic spectra and identifications. Rapid Commun
Mass Spectrom 18: 2162–2168.
66. Eng JK, McCormack AL, Yates Iii JR (1994) An approach to correlate tandem
mass spectral data of peptides with amino acid sequences in a protein database.
Journal of the American Society for Mass Spectrometry 5: 976–989.
67. Dix MM, Simon GM, Cravatt BF (2008) Global mapping of the topography and
magnitude of proteolytic events in apoptosis. Cell 134: 679–691.
68. Yates JR, Cociorva D, Liao L, Zabrouskov V (2006) Performance of a linear ion
trap-Orbitrap hybrid for peptide analysis. Analytic Chemistry 78: 493–500.
Hemolytic Anemia Comparative Proteomics
PLoS ONE | www.plosone.org14April 2012 | Volume 7 | Issue 4 | e34237