Comparative Metaproteomics and Diversity Analysis of Human Intestinal Microbiota Testifies for Its Temporal Stability and Expression of Core Functions

Peer reviewe

Tandem Mass Spectrometry in Resolving Complex Gut Microbiota Functions

This chapter reviews the technological development of high-resolution tandem mass spectrometry (MS) and its capability in analysis of complex microbial systems, particularly in the human gastrointestinal (GI) tract, and presents a few relevant examples of the metaproteomic approach. Protein MS became possible due to the rather simultaneous inventions of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) and is now adopted as a widely used tool in identification as well as characterization of microbiota. These innovations lead to the fast development of proteomics and raised tremendous expectations for the clinical application of proteomic research. The chapter also reviews on studies addressing all the proteins in fecal samples are provided. It describes a more targeted approach that focuses on surface proteins found in intestinal microbiomes. The chapter then focuses on peptide spectral matching (PSM) and de novo sequencing.</p

Osteoclasts secrete osteopontin into resorption lacunae during bone resorption

Abstract Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation

Technical and biological variation in the faecal metaproteomic analysis of subjects A, B and C.

<p>Sample similarities are expressed as Pearson correlations, which were calculated for peptide feature intensities between different sample preparation replicates (experimental), different time point (temporal), and subjects (A, B, C) (ND: not determined; SD: standard deviation; n: number of comparisons). Column “A, B” presents data from an experiment in which the metaproteome of subjects A and B time point one were analysed and column “A, B, C” shows data of a repetition of those samples and an addition of a second time point and subject C. For detailed experimental setup see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029913#s4" target="_blank">Material and Methods</a>” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029913#pone.0029913.s001" target="_blank">Figure S1</a>.</p

The 25 most abundant COGs of the protein core detected in human intestinal samples.

<p>*contribution in percent of specific COG group to the total amount of identified spectra in the core metaproteome.</p><p>COG functional categories: C Energy production and conversion; E Amino acid transport and metabolism; G Carbohydrate transport and metabolism; H Coenzyme transport and metabolism; I Lipid transport and metabolism; J Translation, ribosomal structure and biogenesis; O Posttranslational modification, protein turnover, chaperones.</p