Identification and Validation of Novel Contraction-Regulated Myokines Released from Primary Human Skeletal Muscle Cells

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO₂max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.     

Muscle tissue as an endocrine organ: comparative secretome profiling of slow-oxidative and fast-glycolytic rat muscle explants and its variation with exercise

The notion that skeletal muscle is a secretory organ capable to release proteins that can act locally in an autocrine/paracrine manner or even in an endocrine manner to communicate with distant tissues has now been recognized. Under this context, a new paradigm has arisen implicating the muscle in metabolism regulation. Considering the evidences that give exercise a protective role against illnesses associated to physical inactivity, it becomes of especial relevance to characterize muscle secreted proteins. In the present study we show for the first time the secretome characterization and the comparative 2-DE secretome analysis among fast-glycolytic (gastrocnemius) and slow-oxidative (soleus) rat muscle explants and its variation after exercise intervention. We have identified 19 differently secreted proteins when comparing soleus and gastrocnemius secretomes, and 10 in gastrocnemius and 17 in soleus distinctive secreted proteins after 1 week of endurance exercise training. Among identified proteins, DJ-1 was found to be more abundant in fast-glycolytic fiber secretomes. On the contrary, FABP-3 was elevated in slow-oxidative fiber secretomes, although its secretion from gastrocnemius muscle increased in exercised animals. These and other secreted proteins identified in this work may be considered as potential myokines.     

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.     

Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training

Regular physical activity protects against several types of diseases. This may involve altered secretion of signaling proteins from skeletal muscle. Our aim was to identify the most abundantly secreted proteins in cultures of human skeletal muscle cells and to monitor their expression in muscles of strength-training individuals. A total of 236 proteins were detected by proteome analysis in medium conditioned by cultured human myotubes, which was narrowed down to identification of 18 classically secreted proteins expressed in skeletal muscle, using the SignalP 3.0 and Human Genome Expression Profile databases together with a published mRNA-based reconstruction of the human skeletal muscle secretome. For 17 of the secreted proteins, expression was confirmed at the mRNA level in cultured human myotubes as well as in biopsies of human skeletal muscles. RT-PCR analyses showed that 15 of the secreted muscle proteins had significantly enhanced mRNA expression in m. vastus lateralis and/or m. trapezius after 11 wk of strength training among healthy volunteers. For example, secreted protein acidic and rich in cysteine, a secretory protein in the membrane fraction of skeletal muscle fibers, was increased 3- and 10-fold in m. vastus lateralis and m. trapezius, respectively. Identification of proteins secreted by skeletal muscle cells in vitro facilitated the discovery of novel responses in skeletal muscles of strength-training individuals.     

Computational reconstruction of the human skeletal muscle secretome

In multicellular organisms, secreted proteins play pivotal regulatory roles in intercellular communication. Proteins secreted by skeletal muscle can act locally on muscle cells through autocrine/paracrine loops and on surrounding tissues such as muscle blood vessels, or they can be released into the blood stream, thus producing systemic effects. By a computational approach, we have screened 6255 products of genes expressed in normal human skeletal muscle. Putatively secreted proteins were identified by sequential steps of sieving, through prediction of signal peptide, recognition of transmembrane regions, and analysis of protein annotation. The resulting putative skeletal muscle secretome consists of 319 proteins, including 78 still uncharacterized proteins. This is the first human skeletal muscle secretome produced by computational analysis. Knowledge of proteins secreted by skeletal muscle could stimulate development of novel treatments for different diseases, including muscle atrophy and dystrophy. In addition, better knowledge of the secretion process in skeletal muscle can be useful for future gene therapy approaches.     

Secreted proteins from adipose tissue and skeletal muscle - adipokines, myokines and adipose/muscle cross-talk

White adipose tissue and skeletal muscle are the largest organs in the body and both are composed of distinct cell types. The signature cell of adipose tissue is the adipocyte while myocytes are the defining cell of skeletal muscle. White adipocytes are major secretory cells and this is increasingly apparent also for myocytes. Both cells secrete a range of bioactive proteins, generally termed adipokines in the case of adipocytes and myokines for muscle cells. There has, however, been some confusion over nomenclature and we suggest that the name myokine is restricted to a protein that is secreted from myocytes, while the term adipokine should be used to describe all proteins secreted from any type of adipocyte (white, brown or brite). These definitions specifically exclude proteins secreted from other cells within adipose tissue and muscle, including macrophages. There is some commonality between the myokines and adipokines in that both groups include inflammation-related proteins - for example, IL-6, Il-8 and MCP-1. Adipokines and myokines appear to be involved in local autocrine/paracrine interactions within adipose tissue and muscle, respectively. They are also involved in an endocrine cross-talk with other tissues, including between adipose tissue and skeletal muscle, and this may be bi-directional. For example, IL-6, secreted from myocytes may stimulate lipolysis in adipose tissue, while adipocyte-derived IL-6 may induce insulin resistance in muscle.     

甘油诱导的骨骼肌损伤修复过程肌间脂形态学及肌分泌因子表达变化

研究发现在甘油诱导的小鼠肌肉损伤修复过程中可能存在肌间脂的沉积,而肌肉分泌因子（myokines）作为特殊的蛋白参与了肌肉与脂肪的多种生理过程.为研究肌肉内注射甘油后对肌间脂生成的影响,以及注射后肌肉分泌因子在肌肉损伤后修复及肌间脂沉积过程中的表达趋势,本文选用三月龄C57BL/6品系小鼠,右腿胫骨前肌注射50% HBSS(V/V）甘油,左腿胫骨前肌注射等量的HBSS缓冲液作为对照.取注射后不同时期小鼠的胫骨前肌,冰冻切片技术检测肌肉再生及肌间脂沉积状况,实时定量PCR检测各分泌因子（IL-6、IL-15、MSTN、FNDC5、FGF21、myonectin和Insl6）的mRNA表达变化,酶联免疫分析（ELISA）检测分泌因子的蛋白表达变化.结果表明,在甘油诱导的肌肉损伤再生修复过程中存在肌间脂的生成,同时IL-6、Insl6、FGF21和IL-15的mRNA相对表达量在肌肉损伤修复过程中的前、中期变化明显,而MSTN和myonectin的mRNA相对表达量则在中、后期变化明显. IL-6、Insl6的蛋白表达量在前、中期明显升高.综上所述,甘油注射可引起肌肉损伤修复,并在这一过程中伴随着肌间脂的沉积,而肌肉分泌因子作为肌肉与脂肪之间的信息交换因子可能参与了肌肉损伤后的再生修复以及肌间脂的形成.     

High level expression and purification of recombinant PEX protein in cultured skeletal muscle cell expression system

Large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications. To achieve high-level expression in eukaryotic cells, the choice of cell line as well as the expression vector is critical. In this report, we demonstrate that a combination of the skeletal muscle cell line, QM7 and a cytomegalovirus promoter-based expression vector can achieve high-level expression of secretory recombinant proteins in eukaryotic cells. We also screened a serum-free medium containing 3 microg/ml insulin suitable for QM7 differentiation and identified a very potent signal peptide from MMP9, which effectively directs secretion of heterologous proteins. The C-terminal hemopexin-like domain of MMP-2, PEX, a powerful candidate for the treatment of diseases associated with neovascularization was expressed in QM7 cells with bioactivity. This skeletal muscle cell-based system may be employed for the production of human proteins of special interests, such as those for structural determination or therapeutical development.     

Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.     

Long-term insulin treatment leads to a change in myosin heavy chain fiber distribution in OLETF rat skeletal muscle

The objective of this study was to investigate molecular and physiological changes in response to long-term insulin glargine treatment in the skeletal muscle of OLETF rats. Male Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats aged 24 weeks were randomly allocated to either treatment with insulin for 24 weeks or no treatment, resulting in three groups. Insulin glargine treatment in OLETF rats (OLETF-G) for 24 weeks resulted in changes in blood glucose levels in intraperitoneal glucose tolerance tests compared with age-matched, untreated OLETF rats (OLETF-C), and the area under the curve was significantly decreased for OLETF-G rats compared with OLETF-C rats (P < 0.05). The protein levels of MHC isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of myosin heavy chain type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (IL-6, IL-15, FNDC5, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats ( P < 0.05). MyoD and myogenin levels were decreased, while IGF-I and GLUT4 levels were increased, in the skeletal muscle of OLETF-G rats ( P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of AMPK, SIRT1, and PGC-1α. Together, our results suggested that changes in the distribution of fiber types by insulin glargine could result in downregulation of myokines and muscle regulatory proteins. The effects were likely associated with activation of the AMPK/SIRT1/PGC-1α signaling pathway. Changes in these proteins may at least partly explain the effect of insulin in skeletal muscle of diabetes mellitus.     

Proteomic Analysis of the Palmitate-induced Myotube Secretome Reveals Involvement of the Annexin A1-Formyl Peptide Receptor 2 (FPR2) Pathway in Insulin Resistance

Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.The obesity epidemic has been linked to the development of metabolic complications such as hyperlipidemia, insulin resistance, and hypertension (1, 2). Hyperlipidemia/dyslipidemia involves abnormally elevated levels of lipids and/or lipoproteins in the plasma (3, 4). Obese patients exhibit characteristics of hyperlipidemia/dyslipidemia, such as abnormal elevations in plasma free fatty acid, cholesterol, and triglyceride levels, as well as a reduction in high-density lipoprotein content (3–5). Elevated free fatty acid levels in the plasma of obese patients play an important role in the development of insulin resistance (6). Hence, lowering the free fatty acid level in plasma has been shown to restore insulin sensitivity in these patients (7). Palmitate (C16:0) is a saturated free fatty acid found in animal plasma. It has been reported that the concentration of plasma palmitate in obese patients is higher than in healthy individuals (6, 8). In molecular studies, palmitate has been found to induce inflammation and insulin resistance in skeletal muscle cells by promoting diacylglycerol accumulation, which in turn activates protein kinase C (PKC)-θ¹ and NF-κB, leading to the inhibition of insulin-stimulated Akt phosphorylation through insulin receptor substrate 1 (IRS1) (S307) phosphorylation and IL-6 secretion (9). Sortilin was recently identified as a mediator of palmitate-dependent insulin resistance, which regulates insulin-induced glucose transporter type 4 (GLUT4) trafficking (10). Therefore, palmitate is an important hyperlipidemic/dyslipidemic component that induces insulin resistance in skeletal muscle cells.Skeletal muscle is thought to function as a tissue that produces and releases cytokines called myokines (11). As part of its extracellular signaling pathway, skeletal muscle secretes myokines that participate in myogenesis, angiogenesis, and nutrient generation in response to factors such as metabolic disorders, including insulin resistance, and exercise (11–13). Some myokines, including IL-6, IL-8, IL-15, and fibroblast growth factor 21, and brain-derived neurotrophic factor (14), are induced by exercise. Although myokines are thought to play a critical role in the regulation of (patho)physiological processes, few studies have investigated the role of myokine in metabolism. Because skeletal muscle has a major role in the regulation of glucose metabolism, it is important to identify putative crucial regulators, secreted from skeletal muscle, that modulate glucose metabolism by acting as autocrine/paracrine mediators as well as endocrine mediators (15).Here, using an optimized secretomics approach, we performed a proteomic analysis of proteins in conditioned media from myotube cultures that were either untreated or treated with palmitate to induce insulin resistance (16, 17). Using a label-free quantitative analysis method, our aim was to characterize the skeletal muscle secretome and to identify skeletal muscle-derived proteins whose secretion is modulated by palmitate-induced insulin resistance. We found 36 putative secretory proteins modulated by palmitate-induced insulin resistance. The secretion of annexin A1 was down-regulated after palmitate treatment, and the annexin A1-formyl peptide receptor 2 (FPR2) pathway played a role in palmitate-induced insulin resistance in skeletal muscle by modulating the PKC-θ pathway.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.     

内生菌KM-1-2全基因组ORFs信号肽和分泌蛋白预测及功能分析

【目的】内生菌普遍存在于植物中,与宿主在长期的进化中形成了互利共生的关系。目前对内生菌和植物之间的互作机制研究较少,为深入了解银杏叶内生菌KM-1-2与寄主植物作用机制,本研究对其分泌蛋白进行预测,并明确其特征。【方法】组合使用信号肽分析软件Signal P,跨膜螺旋结构分析软件TMHMM 2.0和Phobius,蛋白质细胞定位软件PSORT,亚细胞定位软件Target P和GPI锚定位点分析软件big-PI Predictor,预测KM-1-2基因组范围内所有分泌蛋白,定义为分泌组。【结果】KM-1-2全基因组5299条蛋白序列中发现271个具有典型信号肽的分泌蛋白,占全基因组的2.4%;编码这些蛋白的ORF最短为61 bp,最大为2105 bp,平均为373 bp;引导它们的信号肽长度分布在15–37 aa之间,平均为24 aa。信号肽中出现频率最高的氨基酸依次为丙氨酸、亮氨酸和缬氨酸,信号肽切割类型多属于A-X-A型,即SPI切割类型。共66个蛋白质有功能描述,其中包括26个酶类。这些酶主要包括各种糖苷水解酶、酯酶、蛋白酶、碳氧裂解酶等。【结论】通过上述生物信息学分析方法有效实现了银杏叶内生菌KM-1-2分泌蛋白的预测,这些分泌蛋白功能涉及较多的酶类以及其他未知功能,为进一步研究内生菌和植物的互作提供了基础。     

The myokine decorin is regulated by contraction and involved in muscle hypertrophy

The health-promoting effects of regular exercise are well known, and myokines may mediate some of these effects. The small leucine-rich proteoglycan decorin has been described as a myokine for some time. However, its regulation and impact on skeletal muscle has not been investigated in detail. In this study, we report decorin to be differentially expressed and released in response to muscle contraction using different approaches. Decorin is released from contracting human myotubes, and circulating decorin levels are increased in response to acute resistance exercise in humans. Moreover, decorin expression in skeletal muscle is increased in humans and mice after chronic training. Because decorin directly binds myostatin, a potent inhibitor of muscle growth, we investigated a potential function of decorin in the regulation of skeletal muscle growth. In vivo overexpression of decorin in murine skeletal muscle promoted expression of the pro-myogenic factor Mighty, which is negatively regulated by myostatin. We also found Myod1 and follistatin to be increased in response to decorin overexpression. Moreover, muscle-specific ubiquitin ligases atrogin1 and MuRF1, which are involved in atrophic pathways, were reduced by decorin overexpression. In summary, our findings suggest that decorin secreted from myotubes in response to exercise is involved in the regulation of muscle hypertrophy and hence could play a role in exercise-related restructuring processes of skeletal muscle.     

N-glycoproteome Analysis of the Secretome of Human Metastatic Hepatocellular Carcinoma Cell Lines Combining Hydrazide Chemistry,HILIC Enrichment and Mass Spectrometry

Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.     

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.     

Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis

Proteins are released from cells by different secretory pathways. The secretory pathway via the ER-Golgi route - realized by a signal sequence - is referred to as “classical secretion”. In contrast, alternative secretory pathways were summarized as “unconventional protein secretion”. Until now, unconventional protein secretion was lacking attention due to the absence of detailed mechanistic insight and limited experimental access. However, there is a growing number of experimental data showing that a large proportion of secreted proteins is released by these alternative routes. Secretomics - the analysis of all secreted proteins of a cell population - offers the opportunity to gain more functional insight into unconventional protein secretion. Several pitfalls in secretome analysis starting with the analyzed cell model and sample preparation to data analysis have to be considered for detailed characterization of the secretome. Here, we highlight the investigation of secretomes by quantitative LC-MS/MS analysis and discuss pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.