Mass spectrometry-based proteomics of human cerebrospinal fluid. Biomarker discovery and verification in multiple sclerosis

The main focus of this study was to use mass spectrometry-based proteomics to study protein abundance in cerebrospinal fluid (CSF) to reveal proteins that could serve as biomarker candidates in multiple sclerosis (MScl). By combining a CSF pooling strategy and label-free relative quantification we discovered 65 proteins of differential abundance between MScl patients and controls. A selection of 17 biomarker candidates was further subjected to two independent verification steps: using stable isotope dimethyl labeling coupled to Accurate Inclusion Mass Screening (dimethyl-AIMS) and Stable Isotope Dilution Selected Reaction Monitoring (SID-SRM) for targeted quantification. The SID-SRM study included a larger patient cohort of 125 cases and controls. To our knowledge, this is the first report of a larger SRM verification study for biomarker candidates in MScl. The most interesting results from the biomarker discovery and verification study were the significantly decreased abundance of Apolipoprotein D, Cystatin C, Kallikrein-6 and Alpha-1-acid glycoprotein 1 in MScl patients compared to controls. Furthermore, we performed a comprehensive characterization of the normal CSF proteome. We identified 18, 807 peptide mapping to 1987 proteins by applying immuno-affinity depletion and SDS-PAGE for enhanced proteome coverage. We obtain a comprehensive set of reference proteins that could further be used for investigations in MScl. The experiment gave us the opportunity to examine the size distribution on the SDS-PAGE gel of a selection of biomarker candidate proteins in normal CSF, with the aim to reveal potential protein variants (isoforms, truncation products and proteolytic processed products). We hypothesized that the identification of non-tryptic peptides could indicate truncation products of proteins in CSF. 13 and nine non-tryptic peptides were identified for the biomarker candidates Cystatin C and Secretogranin-1, respectively. This information had immediate utility for investigation in MScl. Based on the observed spread in size distribution of biomarker candidate proteins in normal CSF, we aimed to obtain quantitative information of proteins present in both high and low mass fractions on the gel and further target these proteins to obtain an abundance ratio between RRMS (patients) and OIND (controls), and investigate if these ratios differed for the same protein. The most striking observation was the opposite regulation level in CSF of Secretogranin-1.Master i Medisinsk biologiMAMD-MEDBIBMED39

Reprogrammed Cells Display Distinct Proteomic SignaturesAssociated with Colony Morphology Variability

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.publishedVersio

In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.publishedVersio

Leptin receptor signaling regulates protein synthesis pathways and neuronal differentiation in pluripotent stem cells

The role of leptin receptor (OB-R) signaling in linking pluripotency with growth and development and the consequences of dysfunctional leptin signaling on progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying the db/db mutation exhibit metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) show altered expression of proteins involved in embryonic development. An upregulation in expression of eukaryotic translation initiation factor 4e (Eif4e) and Stat3 binding to the Eif4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs toward the neuronal lineage showed defects. Gene editing to correct the point mutation in db/db iPSCs using CRISPR-Cas9, restored expression of neuronal markers and protein synthesis while reversing the metabolic defects. These data imply a direct role for OB-R in regulating metabolism in embryonic fibroblasts and key developmental pathways in iPSCs.publishedVersio

The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation

Bioinformatic analyses of miRNA-mRNA signature during hiPSC differentiation towards insulin-producing cells upon HNF4α mutation

Mutations in the hepatocyte nuclear factor 4α (HNF4α) gene affect prenatal and postnatal pancreas development, being characterized by insulin-producing β-cell dysfunction. Little is known about the cellular and molecular mechanisms leading to β-cell failure as result of HNF4α mutation. In this study, we compared the miRNA profile of differentiating human induced pluripotent stem cells (hiPSC) derived from HNF4α+/Δ mutation carriers and their family control along the differentiation timeline. Moreover, we associated this regulation with the corresponding transcriptome profile to isolate transcript–miRNA partners deregulated in the mutated cells. This study uncovered a steep difference in the miRNA regulation pattern occurring during the posterior foregut to pancreatic endoderm transition, defining early and late differentiation regulatory windows. The pathway analysis of the miRNAome–transcriptome interactions revealed a likely gradual involvement of HNF4α+/Δ mutation in p53-mediated cell cycle arrest, with consequences for the proliferation potential, survival and cell fate acquisition of the differentiating cells. The present study is based on bioinformatics approaches and we expect that, pending further experimental validation, certain miRNAs deregulated in the HNF4α+/Δ cells would prove useful for therapy

Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney one-clip hypertensive rats

Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats

Mapping Proteome Changes in Microsatellite Stable, Recurrent Colon Cancer Reveals a Significant Immune System Signature

Background/Aim: Better stratification of the risk of relapse will help select the right patients for adjuvant treatment and improve targeted therapies for patients with colon cancer. Materials and Methods: To understand why a subset of tumors relapse, we compared the proteome of two groups of patients with colon cancer with similar stage, stratified based on the presence or absence of recurrence. Results: Using tumor biopsies from the primary operation, we identified dissimilarity between recurrent and nonrecurrent mismatch satellite stable colon cancer and found that signaling related to immune activation and inflammation was associated with relapse. Conclusion: Immune modulation may have an effect on mismatch satellite stable colon cancer. At present, immune therapy is offered primarily to microsatellite instable colon cancer. Hopefully, immune therapy in mismatch satellite stable colon cancer beyond PD-1 and PD-L1 inhibitors can be implemented.publishedVersio

Reprogrammed Cells Display Distinct Proteomic SignaturesAssociated with Colony Morphology Variability

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity

The effect of WnT pathway modulators on human iPSC-derived pancreatic beta cell maturation

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation