High-Throughput LC-MS/MS Proteomic Analysis of a Mouse Model of Mesiotemporal Lobe Epilepsy Predicts Microglial Activation Underlying Disease Development.

Uncovering the molecular mechanisms of mesiotemporal lobe epilepsy (MTLE) is critical to identify therapeutic targets. In this study, we performed global protein expression analysis of a kainic acid (KA) MTLE mouse model at various time-points (1, 3, and 30 days post-KA injection -dpi), representing specific stages of the syndrome. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination with label-free protein quantification using three processing approaches for quantification, was applied. Following comparison of KA versus NaCl-injected mice, 22, 53, and 175 proteins were differentially (statistically significant) expressed at 1, 3 and 30dpi, respectively, according to all three quantification approaches. Selected findings were confirmed by multiple reaction monitoring LC-MS/MS. As a positive control, the astrocyte marker GFAP was found to be upregulated (3dpi: 1.9 fold; 30dpi: 12.5 fold), also verified by IHC. The results collectively suggest that impairment in synaptic transmission occurs even right after initial status epilepticus (1dpi), with neurodegeneration becoming more extensive during epileptogenesis (3dpi) and sustained at the chronic phase (30dpi), where also extensive glial- and astrocyte-mediated inflammation is evident. This molecular profile is in line with observed phenotypic changes in human MTLE, providing the basis for future studies on new molecular targets for the disease.

Redox proteomics: from residue modifications to putative biomarker identification by gel- and LC-MS-based approaches.

Quantitative determination of reactive oxygen species and reactive nitrogen species in body fluids, tissues or cells has always been problematic due to their high chemical reactivity and the resulting short half-life. This high reactivity may involve reversible and/or irreversible protein modifications, in particular the covalent oxidative modification of specific amino acid residues. Thus, the occurrence of reactive oxygen species and reactive nitrogen species can be monitored indirectly from the identification of specific protein-chemical footprints. In combination with classical gel-based proteomics or liquid chromatography labeling or label-free techniques, mass spectrometry has emerged as a powerful tool to identify these protein modifications in biological samples. In this review, we present the main methodological approaches for gel-based proteomics and quantitative mass spectrometry applied to oxidative protein modifications, mainly Cys. Representative examples from their application in identifying respective biomarkers in diseases related to oxidative stress are also presented.

Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315-339 for sensitivity to inactivation by 2-sulfonatoethyl methanethiosulfonate (MTSES(-)) and N-ethylmaleimide (NEM) showed a similar pattern: highly sensitive mutants clustering at the motif sequence (323-329) and a short alpha-helical face downstream (332, 333, 336). In the presence of substrate, N325C is protected from alkylation with either MTSES(-) or NEM, whereas sensitivity of A323C to inactivation by NEM is enhanced, shifting IC(50) from 34 to 14 microM. Alkylation or sensitivity of the other mutants is unaffected by substrate; the lack of an effect on Q324C is attributed to gross inability of this mutant for high affinity binding. Site-directed mutants G333R and S336N at the alpha-helical face downstream the motif display specific changes in ligand recognition relative to wild type; G333R allows binding of 7-methyl and 8-methylxanthine, whereas S336N disrupts affinity for 6-thioxanthine. Finally, all assayable motif-mutants are highly accessible to MTSES(-) from the periplasmic side. The data suggest that the NAT motif region lines the solvent- and substrate-accessible inner cavity, Asn-325 is at the binding site, Ala-323 responds to binding with a specific conformational shift, and Gly-333 and Ser-336 form part of the purine permeation pathway.

Cysteine-scanning analysis of helices TM8, TM9a, and TM9b and intervening loops in the YgfO xanthine permease: a carboxyl group is essential at ASP-276.

Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences (259)FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV(314) and (342)TIAVMLVILGLFP(354) including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35-140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10-20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K(i) 79 µm) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC(50) 15 µm) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXGDXXAT) and in TM9a (GXXXDG).

Purification and partial characterization of the xanthine-uric acid transporter (UapA) of Aspergillus nidulans.

UapA, the uric acid-xanthine permease from the filamentous ascomycete Aspergillus nidulans, is one of the most thoroughly characterized purine/H(+) transporters in eukaryotes. Detailed studies have addressed its regulation of expression, at both the transcriptional and post-translational levels, in response to physiological and developmental signals. An extensive kinetic profile towards a plethora of purines and mutational analyses have established models on how UapA recognizes the purine ring and revealed specific amino acid residues involved in proper folding, topogenesis, function and specificity. The present work describes for the first time the purification of the UapA transporter of A. nidulans through overexpression via the strong, ethanol-inducible, glucose-repressible, alcA promoter. Purification, almost to homogeneity, was achieved by Ni(2+) affinity chromatography using a functional His-tagged UapA protein version. It is subsequently shown, by Circular Dichroism (CD) spectroscopy, that the purified protein is structured with a high alpha-helical content, as expected from the in silico predictions. The result of this work opens the way for further, analytical and biochemical studies on UapA at the protein level.

Saliva Proteomics Analysis Offers Insights on Type 1 Diabetes Pathology in a Pediatric Population.

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease.

Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells.

Abstract: Background: Multipotent Mesenchymal Stromal Cells (MSCs) are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS). However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL) from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB) and cord blood (CB) PLs were determined using the Multiple Reaction Monitoring (MRM). Finally, the cell doubling time (CDT), tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

Membrane proteomics of cervical cancer cell lines reveal insights on the process of cervical carcinogenesis.

The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus (HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16+) and HeLa (HPV18+). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including 'HIPPO signaling', 'PI3K/Akt signaling', 'cell cycle: G2/M DNA damage checkpoint regulation' and 'EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets.

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis.

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

SRM/MRM targeted proteomics as a tool for biomarker validation and absolute quantification in human urine.

Urine has always been one of the most suitable body fluids for clinical applications. Absolute quantification of disease protein biomarkers in body fluids such as urine is a key step in the biomarker development pipeline. Nevertheless, identification of groups of proteins in complex biological samples is challenging. Traditional affinity-based methodologies such as ELISA are used to verify the presence of biomarkers in clinical samples. More recently, targeted mass spectrometry-based strategies have been developed for biomarker validation, offering an alternative. The great advantage of targeted mass spectrometry-based methodologies is that they allow accurate and specific simultaneous quantification of several biomarkers (multiplexing). Peptides are used as protein surrogates, measured using triple quadrupole instruments in selected reaction monitoring/multiple reaction monitoring mode. In this review, the workflow of selected reaction monitoring/multiple reaction monitoring for disease biomarker validation in urine is presented and assay performance in the latest studies is described.

Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics.

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease-associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE-MS), has been increasingly used for the detection of peptide biomarkers (<20 kDa) in body fluids such as urine. This review presents the most relevant urinary proteomic studies addressing the application of CZE-MS in clinically relevant biomarker research between the years 2006 and 2014.

Comparison of higher energy collisional dissociation and collision-induced dissociation MS/MS sequencing methods for identification of naturally occurring peptides in human urine.

PURPOSE: The aim of this study is to determine the best fragmentation method for sequence identification of naturally occurring urinary peptides in the field of clinical proteomics. EXPERIMENTAL DESIGN: We used LC-MS/MS analysis of urine samples to determine the analytical performance of higher energy collisional dissociation (HCD), CID with high and low resolution MS/MS for the identification of naturally occurring peptides in the low molecular weight urinary proteome. RESULTS: HCD and CID high-resolution generated a 22% error rate in peptide sequence identifications. CID low-resolution showed significantly higher error rates (37%). Excluding the error rate (i.e rejection of cysteine-containing peptides), we observed a higher degree of overlap between HCD and CID high resolution for identification of peptide sequences of rank 1 and cross-correlation ≥ 1.9 (262 peptide sequences) compared to CID low (208 peptide sequences with HCD and 192 peptide sequences with CID high). Reproducibility of detected peptides in three out of the five replicates was also higher in HCD and CID high in relation to CID low resolution. CONCLUSION AND CLINICAL RELEVANCE: Our data demonstrated that HCD and CID high-resolution performed with better accuracy and reproducibility than CID low resolution in respect to the identification of naturally occurring urinary peptide sequences.

Mass spectrometry-based membrane proteomics in cancer biomarker discovery.

Membrane proteins are involved in central processes such as cell signaling, cell-cell interactions and communication, ion and metabolite transport and in general play a crucial role in cell homeostasis. Cancer and cancer metastasis have been correlated to protein expression levels and dysfunction, with membrane proteins playing an important role, and are thus used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Despite the critical biological significance of membrane proteins, proteomic analysis has been a challenging task due to their hydrophobicity. In this review, recent advances in the proteomic analysis of membrane proteins are presented, focusing on membrane fraction enrichment techniques combined with labeled or label-free shotgun proteomics approaches for the identification of potential cancer biomarkers.