Expression profiles of spots showing up- or down-regulation in patient samples.

<p>Of all spots included in the analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034237#pone-0034237-t002" target="_blank">Table 2</a>), 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.</p

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 <b>Table 3</b>).

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034237#pone-0034237-t003" target="_blank">Table 3</a>.</p

Sample characterization (Blood Parameters and Enzyme Assays).

**<p>blood transfusion prior to drawing blood: HA09 42 days, HA24 37 days.</p><p>m: male.</p><p>f: female.</p><p>UI: International Unit.</p><p>G6PD: Glucose -6- Phosphate Dehydrogenase.</p><p>TPI: Triose Phosphate Isomerase.</p><p>GPI: Glucose Phosphate Isomerase.</p><p>ND: No Data.</p><p>The complete panel of enzyme assays performed on all samples in this study including controls can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034237#pone-0034237-t001" target="_blank">Table 1</a>.</p

Principal Component Analysis was calculated by Same Spots software.

<p>Dot plot of all spots included in analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034237#pone-0034237-t002" target="_blank">Table 2</a>) 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.</p

Summary of spots detected by Same Spots Software.

<p>Total number: Number of spots detected by Same Spots Software after filtering (area size and minimum volume).</p><p>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.</p><p>Excluded: Number of spots that were excluded.</p><p>Included: Number of “Differentially expressed” minus “Excluded”.</p><p>Picked: Number of spots picked and trypsinized for mass spec analysis.</p><p>w/o ID: Number of spots where no peptide could be identified.</p><p>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.</p

Expression profiles of spots predominantly containing Chaperone proteins are shown (see also Supplementary Table S6).

<p>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.</p

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.

<p>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.</p

Exportin 7, Fumarate Hydratase and Purine Nucleoside Phosphorylase protein expression.

<p>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.</p

Proteins with parental expression intermediate between patient and unrelated control samples.

<p>ID: Name of sample set (HA09, HA19, HA21, HA24).</p><p>Spot rank: Rank of spot as assigned by Same Spots Software depending on fold change (normalized volume) comparing the highest to lowest sample,</p><p>Fold Change: Comparison of normalized volume in Patient sample with average of Control and Standard sample.</p><p>Gene: HGNC Symbol for coding human gene.</p><p>Protein: HGNC Symbol for protein identified.</p

Expression profiles of proteins involved in protein degradation (see also Supplementary Table S7).

<p>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).</p