Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 μg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.

No abstract

To identify markers in the CSF of multiple sclerosis (MS) subtypes, we used a two-step proteomic approach: (i) Discovery proteomics compared 169 pooled CSF from MS subtypes and inflammatory/degenerative CNS diseases (NMO spectrum and Alzheimer disease) and healthy controls. (ii) Next, 299 proteins selected by comprehensive statistics were quantified in 170 individual CSF samples. (iii) Genes of the identified proteins were also screened among transcripts in 73 MS brain lesions compared to 25 control brains. F-test based feature selection resulted in 8 proteins differentiating the MS subtypes, and secondary progressive (SP)MS was the most different also from controls. Genes of 7 out these 8 proteins were present in MS brain lesions: GOLM was significantly differentially expressed in active, chronic active, inactive and remyelinating lesions, FRZB in active and chronic active lesions, and SELENBP1 in inactive lesions. Volcano maps of normalized proteins in the different disease groups also indicated the highest amount of altered proteins in SPMS. Apolipoprotein C-I, apolipoprotein A-II, augurin, receptor-type tyrosine-protein phosphatase gamma, and trypsin-1 were upregulated in the CSF of MS subtypes compared to controls. This CSF profile and associated brain lesion spectrum highlight non-inflammatory mechanisms in differentiating CNS diseases and MS subtypes and the uniqueness of SPMS.

Neuroinflammation is a hallmark of Alzheimer's disease and TNFα as the main inducer of neuroinflammation has neurodegenerative but also pro-regenerative properties, however, the dose-dependent molecular changes on signaling pathway level are not fully understood. We performed quantitative proteomics and phospho-proteomics to target this point.In HT22 cells, we found that TNFα reduced mitochondrial signaling and inhibited mTOR protein translation signaling but also led to induction of neuroprotective MAPK-CREB signaling. Stimulation of human neurons with TNFα revealed similar cellular mechanisms. Moreover, a number of synaptic plasticity-associated genes were altered in their expression profile including CREB.SiRNA-mediated knockdown of CREB in human neurons prior to TNFα stimulation led to a reduced number of protein/phospho-protein hits compared to siRNA-mediated knockdown of CREB or TNFα stimulation alone and countermeasured the reduced CREB signaling. In vivo data of TNFα knockout mice showed that learning ability did not depend on TNFα per se but that TNFα was essential for preserving the learning ability after episodes of lipopolysaccharide-induced neuroinflammation. This may be based on modulation of CREB/CREB signaling as revealed by the in vitro / in vivo data.Our data show that several molecular targets and signaling pathways induced by TNFα in neurons resemble those seen in Alzheimer's disease pathology.

Background: Multiple sclerosis (MS) is characterized by different degree of inflammatory and neurodegenerative features in the early relapsing vs. progressive subtypes. By using controls with different extent of inflammation vs. neurodegeneration, we examined the CSF proteome to identify molecular markers that differentiate between subtypes of MS. Gene expression of specific proteins were explored in MS brain lesions with diverse pathological background. Methods: (i) First, we compared the proteome by LC-MS/MS in 169 pooled CSF from MS subtypes to inflammatory/degenerative controls: AQP4-IgG-positive and AQP4-IgG-negative neuromyelitis optica spectrum disorder (NMOSD), Alzheimer’s disease (AD), and healthy controls. F-test based feature selection was used to cluster diseases and MS subtypes. (ii) Next, we selected 299 molecules by comprehensive statistics, and quantified them in the individual CSF samples. (iii) We also screened the genes of MS-specific CSF proteins in transcriptomes of 73 MS brain lesions with different pathology. Results: We identified 11 proteins that separated diseases, and 8 proteins that clustered MS subtypes. Secondary progressive (SP)MS had the most unique proteome characterized by upregulation of intrinsic pathway proteins of the coagulation pathway. SPMS also clustered far from NMOSD indicating less inflammatory pathways. Primary progressive (PP)MS was more similar to relapsing-remitting (RR)MS than SPMS. Quantification of 299 proteins in 170 individual CSF samples identified 5 molecules uniquely upregulated in MS subtypes and in AQP4-IgG-positive NMOSD, respectively. Chitinase-3-like protein 1 (CHI3L1) was upregulated in part of PPMS and remission CSF samples, and it was expressed by astrocytes in chronic active lesions. GFAP was upregulated in 70% of AQP4-IgG-positive NMOSD but only in 40% of AQP4-IgG-negative NMOSD. Conclusions: By the combination of untargeted and targeted quantitative analysis, we identified CSF molecular markers of axonal growth inhibition, lipid binding, and protein/lipid transport that differentiated between neuroinflammatory and neurodegenerative diseases, and also MS subtypes. The majority of them were expressed in MS brain lesions suggesting their origin from the brain tissue and not from the systemic compartment. Data suggest that the CSF proteome of SPMS is different from PPMS, and astrocyte damage may not be major pathology in part of the AQP4-IgG seronegative NMOSD.