Targeted label‐free quantification of interleukin‐8 in PMA‐activated U937 cell secretome by nanoLC‐ESI‐MS/MS‐sSRM

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12‐myristate 13‐acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin‐8 (IL‐8). Next, a label‐free nanoLC‐ESI‐MS/MS‐sSRM method for quantification of IL‐8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM‐MS using one proteotypic peptide as precursor ion and four mass transitions. Label‐free quantification was performed by external calibration using IL‐8 standard. Validation results indicated that the method was suited for the quantification of IL‐8 in the secretome. The maximal IL‐8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL‐8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.     

Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry

Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.     

The Botrytis cinerea early secretome

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.     

Quantitative in-depth analysis of the dynamic secretome of activated Jurkat T-cells

Proteins secreted by cells are of the highest biomedical relevance since they play a significant role in the progression of numerous diseases. However, characterization of the proteins specifically secreted in response to precise stimuli is challenging, since these proteins are contaminated by cellular byproducts. Here we present a method to characterize a dynamic secretome and demonstrate its utility by performing the deepest quantitative analysis to date of proteins secreted by lymphoid Jurkat T-cells upon activation. Cell-free supernatant proteins were analyzed by using an optimized protocol for differential (18)O/(16)O-labeling and LC-MS/MS, followed by statistical analysis using a random-effects model. More than 4000 unique peptides belonging to 1288 proteins were identified and a large proportion could be quantified. To determine the proteins whose secretion was up-regulated upon T-cell activation, protein variance of the null hypothesis was estimated after protein classification in terms of secretion and ontology using bioinformatic tools. 62 proteins showed a statistically significant change in abundance upon cell activation and most of them (49 proteins) were up-regulated. These proteins were functionally involved mainly in inflammatory response, signal transduction, cell growth and differentiation and cell redox homeostasis. Our approach provides a promising technology for the high-throughput quantitative study of dynamic secretomes.     

Profiling of the human monocytic cell secretome by quantitative label-free mass spectrometry identifies stimulus-specific cytokines and proinflammatory proteins

The immune response to pathogens or injury relies on the concerted release of cytokines and proteins with biological activity important for host protection, host defense, and wound healing. Consequently, the secretome of immune cells provides a promising resource for discovery of specific molecular markers and targets for pharmacological intervention. Here, we employ label-free MS for unbiased, quantitative profiling of the human monocytic cell secretome under different proinflammatory stimuli. The quantitative secretome profiles reveal the highly stimulus-dependent cellular response and differential, specific secretion of more than 200 proteins, including important proinflammatory proteins and cytokines.     

Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.     

Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip‐based,high pH,reversed‐phase fractionation

Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole‐cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte–neuron communication. To build a reference proteome, we established a C8‐D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two‐step digestion, filter‐aided sample preparation, StageTip‐based high pH fractionation, and high‐resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High‐confidence whole‐cell proteomes and secretomes are valuable resources in studying astrocyte function by label‐free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 ( http://proteomecentral.proteomexchange.org/dataset/PXD000501 ).     

In‐depth characterization of the secretome of mouse CNS cell lines by LC‐MS/MS without prefractionation

Microglia, astrocytes, and neurons, which have important functions in the central nervous system (CNS), communicate mutually to generate a signal through secreted proteins or small molecules, but many of which have not been identified. Because establishing a reference for the secreted proteins from CNS cells could be invaluable in examining cell‐to‐cell communication in the brain, we analyzed the secretome of three murine CNS cell lines without prefractionation by high‐resolution mass spectrometry. In this study, 2795 proteins were identified from conditioned media of the three cell lines, and 2125 proteins were annotated as secreted proteins by bioinformatics analysis. Further, approximately 500 secreted proteins were quantifiable as differentially expressed proteins by label‐free quantitation. As a result, our secretome references are useful datasets for the future study of neuronal diseases. All MS data have been deposited in the ProteomeXchange with identifier PXD001597 ( http://proteomecentral.proteomexchange.org/dataset/PXD001597 ).     

Stem cells: Insights into the secretome

Stem cells have been considered as possible therapeutic vehicles for different health related problems such as cardiovascular and neurodegenerative diseases and cancer. Secreted molecules are key mediators in cell–cell interactions and influence the cross talk with the surrounding tissues. There is strong evidence supporting that crucial cellular functions such as proliferation, differentiation, communication and migration are strictly regulated from the cell secretome. The investigation of stem cell secretome is accumulating continuously increasing interest given the potential use of these cells in regenerative medicine. The scope of the review is to report the main findings from the investigation of stem cell secretome by the use of contemporary proteomics methods and discuss the current status of research in the field. This article is part of a Special Issue entitled: An Updated Secretome.     

Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.     

Mining of serum glycoproteins by an indirect approach using cell line secretome

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experi-mental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.     

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label‐free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.     

Approaches to the study of the cell secretome

The secretome, or secretomics, has recently emerged as a new term to describe the global study of proteins that are secreted by a cell, tissue or organism at any given time or under certain conditions. The secretome constitutes an important class of proteins that control and regulate a multitude of biological and physiological processes, thus making it a clinically relevant source for biomarkers and therapeutic target discoveries. There are several approaches that are being implemented to study such a class of proteins; however, each of these approaches has its advantages and limitations. While genome-wide studies using signal predictions can provide a comprehensive analysis of the secretome, the detection and quantification of the actual secreted proteins in a tissue would be more relevant. The goal of this review is to provide an overview of the methods currently used to analyze such a class of proteins, as well as the challenges encountered during the study of the secretome. The implication of studying the cell secretome together with its clinical relevance will be also covered.     

Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.     

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display

In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high‐throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL‐1 on neutrophils and thereby inhibits the interaction between PSGL‐1 and P‐selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti‐cancer therapy. Our findings provide new insights into the pathogenesis of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.     

Mass spectrometric and computational analysis of cytokine‐induced alterations in the astrocyte secretome

The roles of astrocytes in the CNS have been expanding beyond the long held view of providing passive, supportive functions. Recent evidence has identified roles in neuronal development, extracellular matrix maintenance, and response to inflammatory challenges. Therefore, insights into astrocyte secretion are critically important for understanding physiological responses and pathological mechanisms in CNS diseases. Primary astrocyte cultures were treated with inflammatory cytokines for either a short (1 day) or sustained (7 days) exposure. Increased interleukin‐6 secretion, nitric oxide production, cyclooxygenase‐2 activation, and nerve growth factor (NGF) secretion confirmed the astrocytic response to cytokine treatment. MS/MS analysis, computational prediction algorithms, and functional classification were used to compare the astrocyte protein secretome from control and cytokine‐exposed cultures. In total, 169 secreted proteins were identified, including both classically and nonconventionally secreted proteins that comprised components of the extracellular matrix and enzymes involved in processing of glycoproteins and glycosaminoglycans. Twelve proteins were detected exclusively in the secretome from cytokine‐treated astrocytes, including matrix metalloproteinase‐3 (MMP‐3) and members of the chemokine ligand family. This compilation of secreted proteins provides a framework for identifying factors that influence the biochemical environment of the nervous system, regulate development, construct extracellular matrices, and coordinate the nervous system response to inflammation.     

Comparative secretome analysis of human bone marrow‐derived mesenchymal stem cells during osteogenesis

Osteogenesis is a tightly regulated process that involves coordinated extracellular signals from autocrine and paracrine loops. Secretory proteins during osteogenesis can inhibit cell proliferation and activate cell differentiation toward mature osteoblasts, which are characterized by mineralization. In this study, we attempted to identify these secretory proteins during osteogenesis using LC–MS/MS analysis. We compared the secretome between undifferentiated human bone marrow‐derived mesenchymal stem cells (hBMSCs) and differentiated osteoblasts. Among 315 proteins that were identified, 177 proteins were present at increased levels in osteoblasts, whereas 88 proteins were present at decreased levels. Among the identified proteins, several were validated by quantitative RT‐PCR and immunoblot analysis. Of particular interest, calcium homeostasis‐related proteins were upregulated, whereas stem cell proliferation‐related proteins and other lineage‐related proteins were downregulated during osteogenesis. These findings provide information about the dynamic changes in the expression and secretion of proteins during osteogenesis and suggest the putative role of secretory proteins in osteogenesis. J. Cell. Physiol. 228: 216–224, 2013. © 2012 Wiley Periodicals, Inc.