Extracellular vesicles from pristane-treated CD38-deficient mice express an anti-inflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice.

In CD38-deficient ( Cd38-/- ) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-to-mesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1-enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristane-treated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Distinct serum proteome profiles associated with collagen-induced arthritis and complete Freund's adjuvant-induced inflammation in CD38⁻/⁻ mice: The discriminative power of protein species or proteoforms.

Collagen-type-II-induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38(-/-) than in wild-type (WT) mice. ProteoMiner-equalized serum samples were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38(-/-) versus WT mice either with arthritis (CIA(+) ), with no arthritis (CIA(-) ), or with inflammation (complete Freund's adjuvant (CFA)-treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA(+) from CIA(-) mice, and WT from CD38(-/-) mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA(+) CD38(-/-) mice from CIA(+) WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38(-/-) and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low-abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 (http://proteomecentral.proteomexchange.org/dataset/PXD001788, http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071).

Cluster-based comparison of the peptide mass fingerprint obtained by MALDI-TOF mass spectrometry. A case study: long-term stability of rituximab.

We evaluated the use of the peptide mass fingerprint (PMF) obtained by matrix assisted laser desorption and ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) to track changes in the structure of a protein. The first problem we had to overcome was the inherent complexity of the PMF, which makes it difficult to compare. We dealt with this problem by developing a cluster-based comparison algorithm which takes into account the proportional error made by the mass spectrometer. This procedure involves grouping together similar masses in an intelligent manner, so that we can determine which data correspond to the same peptide (any slight differences can be explained as experimental errors), and which of them are too different and thus more likely to represent different peptides. The proposed algorithm was applied to track changes in a commercially available monoclonal antibody (mAb), namely rituximab (RTX), prepared under the usual hospital conditions and stored refrigerated (4 °C) and frozen (-20 °C) for a long term study. PMFs were obtained periodically over three months. For each checked time, five replicates of the PMFs were obtained in order to evaluate the similarities between them by means of the occurrences of the particular peptides (m/z). After applying the algorithm to the PMF, different approaches were used to analyse the results. Surprisingly, all of them suggested that there were no differences between the two storage conditions tested, i.e. the RTX samples were almost equally well preserved when stored refrigerated at 4 °C or frozen at -20 °C. The cluster-based methodology is new in protein mass spectrometry and could be useful as an easy test for major changes in proteins and biopharmaceutics for diverse applications in industry and other fields, and could provide additional stability data in relation to the practical use of anticancer drugs.

Increased expression and phosphorylation of the two S100A9 isoforms in mononuclear cells from patients with systemic lupus erythematosus: a proteomic signature for circulating low-density granulocytes.

Proteins differentially expressed in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients versus Normal controls were identified by 2-DE and MALDI-MS. Thus, S100A9 expression was significantly increased in SLE PBMCs relative to Normal PBMCs at both mRNA and protein levels. Increased S100A9 levels in SLE PBMCs correlated positively with the abnormal presence of low-density granulocytes (LDGs) detected by flow-cytometry in the mononuclear cell fractions. Another set of proteins that were differentially expressed in SLE PBMCs formed S100A9-independent clusters, suggesting that these differences in protein expression are in fact reflecting changes in the abundance of specific cell types. In SLE PBMCs spots of the two S100A9 isoforms, S100A9-l and S100A9-s, and their phosphorylated counterparts were identified and confirmed to be phosphorylated at Thr(113) by MS/MS analyses. In addition, the phorbol ester PMA alone or in combination with ionomycin induced a stronger increase in threonine phosphorylation of S100A9 in SLE than in Normal PBMCs, while the same stimuli caused the opposite effect on phosphorylation and activation of Erk1/2, suggesting the existence of an abnormal S100A9 signaling in SLE PBMCs. Therefore, the expansion and activation of LDGs in SLE seems to underlie this prominent S100A9 signature.

Proteomic analysis of plasma from patients with systemic lupus erythematosus: increased presence of haptoglobin alpha2 polypeptide chains over the alpha1 isoforms.

In the present study plasma samples from 15 systemic lupus erythematosus (SLE) patients and 16 healthy controls of initially unknown haptoglobin (Hp) phenotype were separated by 2-DE, and tryptic digests of the excised Hpalpha polypeptide chain spots were analyzed by MALDI-TOF-MS. Selected tryptic peptides were sequenced by nano-(n)ESI-IT MS/MS. The six major Hp phenotypes were present, although with distinct frequencies in controls and SLE patients. Thus, there were an increased proportion of SLE patients with Hp 2-2, or Hp 2-1S phenotypes. The Hp phenotype distribution resulted in allele frequencies of 0 625 (Hp(2)), 0.281 (Hp(1S)), and 0.093 (Hp(1F)) in healthy controls, correlating fairly well with the allele frequencies of European populations. In contrast, the Hp allele frequencies of the SLE patients were 0.733 (Hp(2)), 0.233 (Hp(1S)), and 0.033 (Hp1(1F)), which clearly indicated an increased frequency of Hp(2), a similar proportion of Hp(1S) and a diminished proportion of Hp(1F) in SLE patients compared with that in healthy controls. Preferential Hpalpha2 expression in SLE patients may contribute to some of the clinical manifestations of the disease such as hypergammaglobulinemia, systemic vasculitis, and cardiovascular disorders.