Differential Proteomic Analysis of Complex Mixtures by Label-Free nLC MS/MS.

After over two decades of constant evolution, proteomics can be truly considered nowadays as a high-throughput technique. Latest advances performed in sample preparation, instrumentation, and data analysis tools enable proteome-wide detection and quantification of proteins in complex samples.Label-free quantification by nanoscale liquid chromatography coupled online to tandem mass spectrometry (nLC MS /MS ) is a straightforward procedure for relative protein quantification. This approach allows to get deeper insights of what molecular changes are involved in the biological system we want to study in an unbiased manner.This chapter describes methods for sample preparation prior to mass spectrometry analysis. Besides, we describe a standard acquisition method, and some common bioinformatics analyses that help extracting biologically relevant information out of the achieved data.

Molecular Histology Analysis of Cryopreserved Tissue Using Peptide/Protein MALDI-TOF Imaging Mass Spectrometry (MALDI-IMS).

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has emerged as a powerful tool for analyzing the spatial distribution of peptides, small proteins, and other molecules within biological tissues. The obtained signals can be correlated with underlying tissue architecture, without any geometrical distortion, enabling the so-called molecular histology. Here, we analyzed cryopreserved tissue samples employing the MALDI-IMS for proteins and peptides. We used a nonstandard OCT-free cryo-slicing protocol, followed by Carnoy delipidation. Automated matrix spray was utilized to circumvent some of MALDI-IMS technology drawbacks in protein and peptide analysis.

Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed.

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

Borrelia burgdorferi infection induces long-term memory-like responses in macrophages with tissue-wide consequences in the heart.

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.

In-depth proteomics and natural peptidomics analyses reveal antibacterial peptides in human endometrial fluid.

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR < 1%, of which 2261 were found in >50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SIGNIFICANCE: The endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

Regulation of macrophage activity by surface receptors contained within Borrelia burgdorferi-enriched phagosomal fractions.

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi.

Differential proteomic analysis of endometrial fluid suggests increased inflammation and impaired glucose metabolism in non-implantative IVF cycles and pinpoints PYGB as a putative implantation marker.

STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. STUDY DESIGN, SIZE, DURATION: We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. LIMITATIONS, REASONS FOR CAUTION: Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S): This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Mass Spectrometric Identification of Endogenous Peptides.

Peptidomics is an emerging field focused in the analysis of endogenous peptides. Naturally occurring peptides are often endogenously produced protein fragments. Cleavage of precursor proteins by proteases generates peptides that may gain specialized functions not ascribed to their precursors, and which could reflect the state of a cell under certain physiological conditions or pathological processes.Since peptides are found in complex matrices (e.g., serum, tear, urine, cerebrospinal fluid), they need to be isolated from the matrix and concentrated before they can be analyzed on mass spectrometry. This chapter describes methods for sample preparation prior to mass spectrometry analysis. In addition, different peptide fragmentation techniques are described which are complementary when analyzing naturally occurring peptides by liquid chromatography coupled online to tandem mass spectrometry.

TUBEs-Mass Spectrometry for Identification and Analysis of the Ubiquitin-Proteome.

Mass spectrometry (MS) has become the method of choice for the large-scale analysis of protein ubiquitylation. There exist a number of proposed methods for mapping ubiquitin sites, each with different pros and cons. We present here a protocol for the MS analysis of the ubiquitin-proteome captured by TUBEs and subsequent data analysis. Using dedicated software and algorithms, specific information on the presence of ubiquitylated peptides can be obtained from the MS search results. In addition, a quantitative and functional analysis of the ubiquitylated proteins and their interacting partners helps to unravel the biological and molecular processes they are involved in.

MALDI-Imaging Mass Spectrometry: a step forward in the anatomopathological characterization of stenotic aortic valve tissue.

Aortic stenosis (AS) is the most common form of valve disease. Once symptoms develop, there is an inexorable deterioration with a poor prognosis; currently there are no therapies capable of modifying disease progression, and aortic valve replacement is the only available treatment. Our goal is to study the progression of calcification by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) and get new insights at molecular level that could help in the understanding of this disease. In this work, we analyzed consecutive slices from aortic valve tissue by MALDI-IMS, to establish the spatial distribution of proteins and peptides directly from the surface of the histological sections. The analysis showed different structures corresponding to regions observed in conventional histology, including large calcification areas and zones rich in collagen and elastic fibers. Peptide extraction from the tissue, followed by liquid chromatography mass spectrometry analysis, provided the identification of collagen VI α-3 and NDRG2 proteins which correlated with the masses obtained by MALDI-IMS and were confirmed by immunohistochemistry. These results highlighted the molecular mechanism implied in AS using MALDI-IMS, a novel technique never used before in this pathology. In addition, we can define specific regions proving a complementary resolution of the molecular histology.