Protocadherin 7–Associated Membranous Nephropathy

BackgroundMembranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%–90% of target antigens in MN.MethodsWe performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue.ResultsMS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7.ConclusionsMN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.

Potential urinary aging markers of 20-month-old rats

Urine is a very good source for biomarker discovery because it accumulates changes in the body. However, a major challenge in urinary biomarker discovery is the fact that the urinary proteome is influenced by various elements. To circumvent these problems, simpler systems, such as animal models, can be used to establish associations between physiological or pathological conditions and alterations in the urinary proteome. In this study, the urinary proteomes of young (two months old) and old rats (20 months old; nine in each group) were analyzed using LC-MS/MS and quantified using the Progenesis LC-MS software. A total of 371 proteins were identified, 194 of which were shared between the young and old rats. Based on criteria of a fold change ≥2,P< 0.05 and identification in each rat of the high-abundance group, 33 proteins were found to be changed (15 increased and 18 decreased in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), eight proteins showed consistent changes in all rats of the groups; two of these proteins are also altered in the urinary proteome of aging humans. However, no shared proteins between our results and the previous aging plasma proteome were identified. Twenty of the 33 (60%) altered proteins have been reported to be disease biomarkers, suggesting that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs which, according to the Human Protein Atlas, are strongly expressed in the kidney, intestine, cerebellum and lung. Therefore, the urinary proteome may reflect aging conditions in these organs.

The potential urinary aging markers of 20-month-old rats

Urine is a very good source for biomarker discovery because it accumulates the changes of body. The urinary proteome is influenced by various factors, which is a major challenge in urinary biomarker discovery. To circumvent these problems, simpler systems, such as animal models, should be used to establish associations between physiological or pathological conditions and changes in the urinary proteome. In this study, the urinary proteome of young (2-month-old) and old rats (20-month-old; 9 in each group) were analyzed using LC-MS/MS and quantified using the Progenesis LC-MS software. A total of 371 proteins were identified, 194 of which were shared between young and old rats. Based on the criteria of a fold change ≥ 2, P < 0.05 and being identified in each rat in the high abundance group, 33 proteins were changed (15 up-regulated and 18 down-regulated in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), 8 proteins were changed consistently in all rats of between the groups, 2 of which are also altered in the urinary proteome of aging humans. There are no shared proteins between our results and the previous aging plasma proteome. Twenty of the 33 (60 %) changed proteins have been reported to be disease biomarkers, which implies that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs, which are strongly expressed in the kidney, intestine, cerebellum and lung, according to the human protein ATLAS. Therefore, the urinary proteome may reflect aging conditions in these organs.

The potential urinary aging markers of 20-month-old rats

Urine is a very good source for biomarker discovery because it accumulates the changes of body. The urinary proteome is influenced by various factors, which is a major challenge in urinary biomarker discovery. To circumvent these problems, simpler systems, such as animal models, should be used to establish associations between physiological or pathological conditions and changes in the urinary proteome. In this study, the urinary proteome of young (2-month-old) and old rats (20-month-old; 9 in each group) were analyzed using LC-MS/MS and quantified using the Progenesis LC-MS software. A total of 371 proteins were identified, 194 of which were shared between young and old rats. Based on the criteria of a fold change ≥ 2, P < 0.05 and being identified in each rat in the high abundance group, 33 proteins were changed (15 up-regulated and 18 down-regulated in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), 8 proteins were changed consistently in all rats of between the groups, 2 of which are also altered in the urinary proteome of aging humans. There are no shared proteins between our results and the previous aging plasma proteome. Twenty of the 33 (60 %) changed proteins have been reported to be disease biomarkers, which implies that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs, which are strongly expressed in the kidney, intestine, cerebellum and lung, according to the human protein ATLAS. Therefore, the urinary proteome may reflect aging conditions in these organs.

The potential urinary aging markers of 20-month-old rats

Urine is a very good source for biomarker discovery because it accumulates the changes of body. The urinary proteome is influenced by various factors, which is a major challenge in urinary biomarker discovery. To circumvent these problems, simpler systems, such as animal models, should be used to establish associations between physiological or pathological conditions and changes in the urinary proteome. In this study, the urinary proteome of young (2-month-old) and old rats (20-month-old; 9 in each group) were analyzed using LC-MS/MS and quantified using the Progenesis LC-MS software. A total of 371 proteins were identified, 194 of which were shared between young and old rats. Based on the criteria of a fold change ≥ 2, P < 0.05 and being identified in each rat in the high abundance group, 33 proteins were changed (15 up-regulated and 18 down-regulated in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), 8 proteins were changed consistently in all rats of between the groups, 2 of which are also altered in the urinary proteome of aging humans. There are no shared proteins between our results and the previous aging plasma proteome. Twenty of the 33 (60 %) changed proteins have been reported to be disease biomarkers, which implies that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs, which are strongly expressed in the kidney, intestine, cerebellum and lung, according to the human protein ATLAS. Therefore, the urinary proteome may reflect aging conditions in these organs.

Deep Proteomic Profiling Predicts Differential Chemosensitivity In Anaplastic Large Cell Lymphoma Cell Lines

Background The lymphoma proteome is the phenotypic representation of the underlying genetic and epigenetic makeup of each individual patient's tumor. The proteome is rich in druggable targets and offers a unique opportunity for the hematologist to personalize therapy. We developed a deep proteomic profiling method using ion exchange fractionation and tandem mass spectrometry. In this pilot study, we applied this method to detect differentially expressed proteins in anaplastic large cell lymphoma (ALCL) cell lines with previously known (ALK-positive) and unknown (ALK-negative) therapeutic targets. We then asked whether integrative informatic analysis of these data could be used to predict drug sensitivity in each of the cell lines. Methods To examine reproducibility of our method, proteins were extracted independently from 4 pellets each of FE-PD (ALK-negative) and Karpas 299 (ALK-positive) ALCL cells, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Resulting peptides were separated into 6 fractions using strong anion exchange (SAX) chromatography. Peptides in each fraction were analyzed via shotgun proteomics on a QExactive mass spectrometer. Peptide mass spectra (MS/MS) were matched against a RefSeq human protein sequence database using MyriMatch software. Reversed sequences were added to the database to measure identification false discovery rates (FDRs). IDPicker filtered the peptide identifications at 2% FDR. Proteins with at least two unique peptide identifications and five MS/MS matches were considered to be present in the sample. Filtered protein identifications and corresponding spectral counts were used as input to QuasiTel software, which was configured to use proteins with at least one spectrum per biological replicate. Proteins that were significantly differentially expressed (quasi p-value < 0.05) with an absolute log2 fold-change of at least 0.5 fold were loaded into the Ingenuity Pathway Analysis (IPA) software, and a master list of drugs and corresponding gene targets was assembled using PharmGKB database and the Drug-Gene Interaction Database (DGID). The resulting drug-gene target list was merged with the differentially expressed protein identifications. Candidate targets were validated by Western blot and candidate drugs were assessed in viability assays. Results The SAX-LC-MS/MS method identified 10,111 proteins from all replicate analyses of FE-PD and Karpas 299 samples, and 93% of the identified proteome was detected in all 4 replicate analyses. The detected proteome was well represented by key transcription factors, phophatases, kinases, translation regulators and transmembrane regulators. There were 1369 proteins differentially expressed between the 2 cell lines, 709 up regulated in Karpas 299 cell line and 673 up regulated in FE-PD. Differentially expressed proteins also showed consistent expression across the biological replicates (Figure 1). Our integrated approach to identify candidate targets and drugs “rediscovered” ALK in Karpas299 and unexpectedly identified relative overexpression of the IL2-IL2RA-STAT5A-STAT5B network in ALK-negative FE-PD cells. Western blot confirmed these findings. As expected, 50-100 nM crizotinib (ALK inhibitor) decreased Karpas 299 viability (p =0.016) but had no effect on FE-PD (Figure 2). In contrast, 50-100 nM of the experimental STAT5 inhibitor 573108 (EMD Millipore) decreased FE-PD viability (p=0.002) but had no effect on Karpas 299 (Figure 2). Conclusion The lymphoma proteome is complex with 10,000 proteins and contains druggable targets that can be reproducibly identified using SAX-LC-MS/MS. These targets vary across samples and integrated informatic analysis can predict target-drug combinations that have efficacy in an experimental model. These data suggest that SAX-LC-MS/MS could be used to personalize treatment regimens in lymphoma patients. Figure 1: Spectral counts of 1369 differentially expressed proteins between Karpas299 and FE-PD cell lines were normalized and plotted in a heat map. Red color indicates down regulation and yellow color indicates up regulation. Disclosures: No relevant conflicts of interest to declare.

Deciphering the human platelet sheddome

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.