Use Of Proteomics Research Articles

Abstract 219: Identification of the mechanistic target of rapamycin complex 2-associated interactome involved in brain cancer cell motility by affinity purification-mass spectrometry

Abstract The Mechanistic Target of Rapamycin Complex 2 (mTORC2), a multiprotein complex with serine-threonine kinase activity, drives several cellular processes of normal cells to promote proper metabolic activity, survival, proliferation, differentiation and movement. On the other hand, mTORC2 has been implicated as a critical player in tumorigenesis, stimulation of cell growth, cancer metabolic reprogramming, and development of highly migratory and invasive capabilities of cancers. Therefore, characterization of mTORC2 is necessary not only for better understandings of known mTORC2-mediated functions, but also for the discovery of new roles and more interacting partners of mTORC2 signaling network in cancers, particularly ones with hyperactivated mTORC2 such as glioblastoma. However, until now mTORC2 is still partially characterized. Previously, mTORC2 was reported to be associated with Filamin A and Myosin-9 which have been known to play important roles in cellular activities requiring locomotion, including cancer migration and invasion. The complex participates in regulating glioblastoma migration and invasion, and Filamin A acts as a physiological downstream target of mTORC2. Currently, proteomics has emerged as a powerful tool to identify, quantify, and examine a large number of proteins. Therefore, in order to extensively unravel mTORC2 signaling pathway, the use of proteomics is necessary. We characterized mTORC2 by proteomic analysis based on affinity purification coupled to mass spectrometry (AP-MS) using antibodies against RICTOR, a specific component of mTORC2. The protein-protein interactions between RICTOR and other associated proteins were assessed. In addition to canonical components of mTORC2, the study has revealed more insights into a number of mTORC2 interacting partners which can be categorized and related to the main functions previously identified such as actin cytoskeleton reorganization, fatty acid metabolism, protein translation. Interestingly, we also found other types of cytoskeletal proteins, nucleotide metabolism, less-characterized and low abundance proteins associated with mTORC2 such as Hornerin and Desmoplakin. In addition, we have carried out the identification of condition-specific interactions for a dynamic view of mTORC2 interactome and found changes in the mTORC2-associated proteins from different conditions. Furthermore, proteomic approaches can also be relevant to investigation and prediction of mTORC2 upstream regulators, downstream targets, and complex localization. Ultimately, this project will uncover some unknown molecular mechanisms, and result in a significant impact on the field of signal transduction machinery in cancers. Citation Format: Naphat Chantaravisoot, Piriya Wongkongkathep, Fuyuhiko Tamanoi, Trairak Pisitkun. Identification of the mechanistic target of rapamycin complex 2-associated interactome involved in brain cancer cell motility by affinity purification-mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 219. doi:10.1158/1538-7445.AM2017-219

The Use of Proteomics in Assisted Reproduction.

Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally.

Proteomics approaches to investigate cancer radiotherapy outcome: slow train coming

Radiotherapy represents an effective curative strategy that along with surgery and chemotherapy is traditionally used in cancer treatment. An optimal therapy delivers accurate radiation doses to tumour while protecting surrounding normal tissue. Although radiotherapy regimens have improved in recent years, tumour radioresistance and normal tissue toxicity remain major challenges that considerably contribute to the effectiveness of cancer therapy. These factors affect especially radiosensitive individuals in the treatment of whom the balance between effective tumour killing and adverse normal tissue late effects turns out to be problematic. Understanding molecular mechanisms involved in tumour and normal tissue response is essential in order to improve radiotherapy outcome in the field of personalised medicine. Alteration in the global proteome of cells and tissues truthfully reflects the changes in physiology and pathophysiology of biological samples. This makes proteomics analysis a powerful tool principally enabling identification of radioresistance biomarkers in cancer and sensitivity biomarkers in individuals. Yet, the use of proteomics in cancer therapy is still in its infancy and more research is needed to fulfil the great promise of this technique. The present review summarises recent proteomic-based studies searching for biomarkers for more accurate prediction of radiotherapeutic response in tumour and normal tissue.

In search of possible peripheral biomarkers for suicide: Similarities between platelet and cerebrospinal fluid proteome (preliminary results)

Despite the fact that proteomic analysis is becoming widely used in various medical branches its use in psychiatry is still very limited. Majority of psychiatric proteomic research is still oriented mostly on Alzheimer's disease, schizophrenia and depression but very few studies focus on suicidality. We decided, based on the current knowledge, to study suicidal behaviour with the use of proteomics to compare cerebrospinal fluid and platelets. We hypothesized that the same protein group can be detected in pathways that are part of platelet degranulation process in the platelet proteome and cerebrospinal fluid proteome. Based on these findings we suppose, that with use of proteomic analysis a specific protein (group of proteins) can be identified in both, cerebrospinal fluid and platelet proteome in patients with suicidal behavior.Group of proteins identified in our sample in the reactome pathway database (release of platelet secretary granule components and exocytosis of platelet granule contents) supports the idea of link between central nervous system and platelets (“the periphery”). Further research is needed to clarify whether the identified group of proteins taking part in platelet pathways can be used as peripheral biomarkers for suicidal behavior.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Systemic alterations in plasma proteins from women with chronic widespread pain compared to healthy controls: a proteomic study.

Chronic widespread pain (CWP) is a complex pain condition that is difficult to treat. The prevalence of CWP approximates ~10% of the general population, with higher prevalence in women. Lack of understanding of molecular mechanisms has been a challenge for diagnosis and treatment of chronic pain. The aim of this study was to explore the systemic protein changes in CWP compared to those in healthy controls (CON). By applying 2-dimensional gel electrophoresis, we analyzed the protein pattern of plasma samples from women with CWP (n=16) and healthy women (n=23). The proteomic data were analyzed using multivariate statistical models, and altered proteins were identified using mass spectrometry. The proteome analysis was further validated by gel-free Western blot. Multivariate statistical data analysis of quantified proteins revealed 22 altered proteins in women with CWP, compared to CON group. Many of the identified proteins are previously known to be involved in different parts of the complement system and metabolic and inflammatory processes, e.g., complement factor B, vitamin D-binding protein, ceruloplasmin, transthyretin and alpha-2-HS-glycoprotein. These results indicate that important systemic protein differences exist between women with CWP and healthy women. Further, this study illustrates the potential use of proteomics to detect biomarkers that may provide new insights into the molecular mechanism(s) of chronic pain. However, further larger investigations are required in order to confirm these findings before it will be possible to identify proteins as potential pain biomarkers for clinical use.

Potential Use of Proteomics in Shellfish Aquaculture: from Assessment of Environmental Toxicity to Evaluation of Seafood Quality and Safety

Potential Use of Proteomics in Shellfish Aquaculture: from Assessment of Environmental Toxicity to Evaluation of Seafood Quality and Safety

Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications.

Phosphate addition is a posttranslational modification of proteins, and this modification can affect the activity and other properties of intracellular proteins. Different animal species can be used to generate phosphosite-specific antibodies as either polyclonals or monoclonals, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is vital for their use in proteomics and profiling of disease.

Formation of sensor array on the AFM chip surface by magnetron sputtering

Development of atomic force microscopy (AFM)-based nanotechnological approaches to highly sensitive detection of proteins is a perspective direction in biomedical research. These approaches use AFM chips to concentrate the target proteins from the test solution volume (buffer solution, diluted biological fluid) onto the chip surface for their subsequent registration on the chip surface by AFM. Atomic force microscope is a molecular detector that enables protein detection at ultra-low (subfemtomolar) concentrations in single-molecule counting mode. Due to extremely high sensitivity of AFM, its application for multiplexed protein detection is of great interest for use in proteomics and diagnostic applications. In this study, AFM chips containing an array of sensor areas have been fabricated. Magnetron sputtering of chromium and tungsten nanolayers has been used to form optically visible metallic marks on the AFM chip surface to provide necessary precision of AFM probe positioning against each sensor area for scanning. It has been demonstrated that the marks formed by magnetron sputtering of Cr and W are stable on the surface of the AFM chips during the following activation and intensive washing of this surface. The results obtained in our present study allow application of the developed chips for multiplexed protein analysis by AFM.

Application of proteomics in the elucidation of chemical-mediated allergic contact dermatitis.

Allergic contact dermatitis (ACD) is a widespread hypersensitivity reaction of the skin. The cellular mechanisms underlying its development are complex and involve close interaction of different cell types of the immune system. It is this very complexity which has long prevented straightforward replacement of the corresponding regulatory in vivo tests. Recent efforts have already resulted in the development of several in vitro testing alternatives that address key steps of ACD. Yet identification of suitable biomarkers is still a subject of intense research. Search strategies for the latter encompass transcriptomics, proteomics as well as metabolomics approaches. The scope of this review shall be the application and use of proteomics in the context of ACD. This includes highlighting relevant aspects of the molecular and cellular mechanisms underlying ACD, the exploitation of these mechanisms for testing and biomarkers (e.g., in the context of the OECD's adverse outcome pathway initiative) as well as an outlook on emerging proteome targets, for example during the allergen-induced activation of dendritic cells (DCs).

Advancements in Diagnosing Periprosthetic Joint Infections after Total Hip and Knee Arthroplasty.

Periprosthetic joint infection (PJI) is a complication of total joint arthroplasty that is challenging to diagnose. Currently, there is no “gold standard” for definite diagnosis of PJI. A multi-criteria definition has been described for PJI based on microbiology cultures, serum markers, such as erythrocyte sedimentation rate and C-reactive protein (CRP), synovial fluid biomarkers, such as leukocyte esterase and histopathology assessment of the periprosthetic tissue. The conventional serum markers are generally nonspecific and can be elevated in inflammatory conditions. Therefore, they cannot be relied on for definite diagnosis of PJI. Hence, with the use of proteomics, synovial fluid biomarkers such as α-defensin, IL-6, and CRP have been proposed as more accurate biomarkers for PJI. Current methods to culture micro-organisms have several limitations, and can be false-negative and false-positive in a considerable number of cases. In an attempt to improve culture sensitivity, diagnostic methods to target biofilms have recently been studied. The understanding of the concept of biofilms has also allowed for the development of novel techniques for PJI diagnosis, such as visualizing biofilms with fluorescent in-situ hybridization and detection of bacteria via DNA microarray. Lastly, the use of amplification-based molecular techniques has provided methods to identify specific species of bacteria that cause culture-negative PJI. While diagnosing PJI is difficult, these advances could be valuable tools for clinicians.

Assessing the Phagosome Proteome by Quantitative Mass Spectrometry.

Phagocytosis is the process that engulfs particles in vesicles called phagosomes that are trafficked through a series of maturation steps, culminating in the destruction of the internalized cargo. Because phagosomes are in direct contact with the particle and undergo constant fusion and fission events with other organelles, characterization of the phagosomal proteome is a powerful tool to understand mechanisms controlling innate immunity as well as vesicle trafficking. The ability to isolate highly pure phagosomes through the use of latex beads led to an extensive use of proteomics to study phagosomes under different stimuli. Thousands of different proteins have been identified and quantified, revealing new properties and shedding new light on the dynamics and composition of maturing phagosomes and innate immunity mechanisms. In this chapter, we describe how quantitative-based proteomic methods such as label-free, dimethyl labeling or Tandem Mass Tag (TMT) labeling can be applied for the characterization of protein composition and translocation during maturation of phagosomes in macrophages.

A Proteogenomic Approach to Understanding MYC Function in Metastatic Medulloblastoma Tumors.

Brain tumors are the leading cause of cancer-related deaths in children, and medulloblastoma is the most prevalent malignant childhood/pediatric brain tumor. Providing effective treatment for these cancers, with minimal damage to the still-developing brain, remains one of the greatest challenges faced by clinicians. Understanding the diverse events driving tumor formation, maintenance, progression, and recurrence is necessary for identifying novel targeted therapeutics and improving survival of patients with this disease. Genomic copy number alteration data, together with clinical studies, identifies c-MYC amplification as an important risk factor associated with the most aggressive forms of medulloblastoma with marked metastatic potential. Yet despite this, very little is known regarding the impact of such genomic abnormalities upon the functional biology of the tumor cell. We discuss here how recent advances in quantitative proteomic techniques are now providing new insights into the functional biology of these aggressive tumors, as illustrated by the use of proteomics to bridge the gap between the genotype and phenotype in the case of c-MYC-amplified/associated medulloblastoma. These integrated proteogenomic approaches now provide a new platform for understanding cancer biology by providing a functional context to frame genomic abnormalities.

How well can morphology assess cell death modality? A proteomics study.

While the focus of attempts to classify cell death programs has finally shifted in 2010s from microscopy-based morphological characteristics to biochemical assays, more recent discoveries have put the underlying assumptions of many such assays under severe stress, mostly because of the limited specificity of the assays. On the other hand, proteomics can quantitatively measure the abundances of thousands of proteins in a single experiment. Thus proteomics could develop a modern alternative to both semiquantitative morphology assessment as well as single-molecule biochemical assays. Here we tested this hypothesis by analyzing the proteomes of cells dying after been treated with various chemical agents. The most striking finding is that, for a multivariate model based on the proteome changes in three cells lines, the regulation patterns of the 200–500 most abundant proteins typically attributed to household type more accurately reflect that of the proteins directly interacting with the drug than any other protein subset grouped by common function or biological process, including cell death. This is in broad agreement with the 'rigid cell death mechanics' model where drug action mechanism and morphological changes caused by it are bijectively linked. This finding, if confirmed, will open way for a broad use of proteomics in death modality assessment.

Identification of peptides in the terminal ileum of broiler chickens fed diets based on maize and soybean meal using proteomics

A total of 160 Ross PM3 birds were used in a two treatment feeding study in order to explore the usefulness of proteomics to identify the origin of peptides in ileal digesta. Two diets were fed, one conventional maize/soy-based diet acted as a reference whereas a second diet, formulated to be nutritionally equivalent to the reference diet in protein and energy provision, contained 20% raw soy meal in order to (putatively) elicit changes in intestinal protein flow. Each diet was fed to 10 replicate cages of eight birds per cage from Day 1 to 21. Feed and water were available ad libitum and an indigestible marker was included for assessment of ileal digestibility. Weight gain and feed intake were monitored and at the end of the trial period birds were killed, pancreatic mass was measured and the ileum was excised and the contents were collected, immediately frozen in liquid nitrogen and were subsequently lyophilised. Protein from the ileal digesta was extracted and exposed to proteomic analysis with peptide fragments identified and compared with an amalgamated database containing protein sequences from chicken, soy and maize. Addition of 20% raw soy meal to the maize/soy-based diet resulted in a reduction in weight gain, feed intake and an increase in feed conversion ratio (P < 0.001). Pancreatic mass was significantly increased and the apparent ileal digestibility of protein was significantly decreased by raw soy meal inclusion. Overall, a total of 248 proteins were identified from endogenous origin, 336 from soy and 411 from maize. However, the relative abundance of these proteins were ~20–30% for endogenous protein, 65–75% for soy protein and ~2–4% for maize protein. The addition of 20% raw soy meal resulted in an increase in the relative abundance of endogenous protein and a reduction in the relative abundance of protein from soy with no measurable effect on the presence of protein from maize. Specifically, in the endogenous protein fraction, there was a significant reduction in the relative abundance of metalloendopeptidase, aminopeptidase and alkaline phosphatase and a significant increase in the relative abundance of colipase and trypsin, in response to raw soy meal inclusion. For proteins originating from soybean, the addition of raw soy meal to the diet resulted in a significant increase in the relative abundance of protein from the 2S albumin fraction, Kunitz and Bowman–Birk trypsin inhibitors and soybean agglutinin whereas there was a reduction in the relative abundance of globulin and glycinin. Addition of raw soy meal to the diet also resulted in a significant increase in the presence of maize prolamin in the lumen and a significant decrease in the presence of globulin-2, β-1–3-glucanase and cystatin. These results demonstrate considerable potential of proteomics technology to identify changes in the digestion and secretion of protein in the intestine of chickens. Although these data are preliminary and based on an animal model that included diets that were formulated to have a chronic effect on intestinal physiology it is evident that changes in diet composition can have a profound effect on the origin of protein that leaves the ileum.

Abstract 753: The use of proteomics to analyse whole tumors and identify unique stroma cell targets for antibody-based therapeutics

Abstract Solid tumors comprises of two distinct compartments: cancer cells and the stroma that the cancer cells induce and are dispersed in. This stroma contains stromal (Fibroblasts, endothelial and pericyte cells) and infiltrating immune cells (Lymphocytes, macrophages, granulocytes and myeloid derived suppressor cells). Recent oncology research has implicated these stroma cells as promoters of tumor progression and there is thus a strong need to profile the cell membrane proteins present on these cells. For tumors which do not contain infiltrating T cells (non-immunogenic tumors) it has been recognized that it will be necessary to employ therapeutic agents to disrupt the tumor micro-environment (TME); allowing the release and processing of tumor antigens by antigen presenting cells, entry and migration T cells into intrastromal sites and potent activation of T cell responses to tumor antigens. We have conducted in-depth proteomic profiling of tumors from 14 different solid cancer indications with varying degrees of stroma involvement to characterize their immune and stromal cell composition. The data from this work was analyzed in OGAP®; a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify membrane protein expression levels and profiles. It is one of the world's largest proprietary cell-membrane focused proteomic database, with data on over 5000 cancer membrane proteins. Purified stroma cell populations were also profiled to facilitate the interpretation of whole tumor proteomic data. Cell membrane proteins present in tumors from different cancer indications were analyzed by mass spectrometry. We classified tumors as immunogenic or non-immunogenic using validated T cell markers (CD3, CD4, CD8 and CD69). We then profiled the non-immunogenic tumors for fibroblast, macrophage and B cell type markers to try and identify strong stroma cell expression patterns not detected in normal tissues. Importantly, most current antibody therapeutic stroma cell targets (CSF-1R, FAP and VEGF-A) were re-discovered. Several novel stroma cell therapeutic targets were identified which if targeted by a therapeutic antibody have the potential to enhance the T cell immune response in tumors resistant to current immunotherapy. Citation Format: James Ackroyd, Jason Allen, Jo Berry, Lisa Roesch, Martin Barnes, Arnima Bisht, Christian Rohlff, Keith Wilson, Dee Aud, Robert Boyd. The use of proteomics to analyse whole tumors and identify unique stroma cell targets for antibody-based therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 753.

Human Pituitary Adenoma Proteomics: New Progresses and Perspectives.

Pituitary adenoma (PA) is a common intracranial neoplasm that impacts on human health through interfering hypothalamus–pituitary–target organ axis systems. The development of proteomics gives great promises in the clarification of molecular mechanisms of a PA and discovery of effective biomarkers for prediction, prevention, early-stage diagnosis, and treatment for a PA. A great progress in the field of PA proteomics has been made in the past 10 years, including (i) the use of laser-capture microdissection, (ii) proteomics analyses of functional PAs (such as prolactinoma), invasive and non-invasive non-functional pituitary adenomas (NFPAs), protein post-translational modifications such as phosphorylation and tyrosine nitration, NFPA heterogeneity, and hormone isoforms, (iii) the use of protein antibody array, (iv) serum proteomics and peptidomics, (v) the integration of proteomics and other omics data, and (vi) the proposal of multi-parameter systematic strategy for a PA. This review will summarize these progresses of proteomics in PAs, point out the existing drawbacks, propose the future research directions, and address the clinical relevance of PA proteomics data, in order to achieve our long-term goal that is use of proteomics to clarify molecular mechanisms, construct molecular networks, and discover effective biomarkers.

Foodborne pathogens and their toxins

Foodborne pathogens, mostly bacteria and fungi, but also some viruses, prions and protozoa, contaminate food during production and processing, but also during storage and transport before consuming. During their growth these microorganisms can secrete different components, including toxins, into the extracellular environment. Other harmful substances can be also liberated and can contaminate food after disintegration of food pathogens. Some bacterial and fungal toxins can be resistant to inactivation, and can survive harsh treatment during food processing. Many of these molecules are involved in cellular processes and can indicate different mechanisms of pathogenesis of foodborne organisms. More knowledge about food contaminants can also help understand their inactivation. In the present review the use of proteomics, peptidomics and metabolomics, in addition to other foodomic methods for the detection of foodborne pathogenic fungi and bacteria, is overviewed. Furthermore, it is discussed how these techniques can be used for discovering biomarkers for pathogenicity of foodborne pathogens, determining the mechanisms by which they act, and studying their resistance upon inactivation in food of animal and plant origin. Biological significanceComprehensive and comparative view into the genome and proteome of foodborne pathogens of bacterial or fungal origin and foodomic, mostly proteomic, peptidomic and metabolomic investigation of their toxin production and their mechanism of action is necessary in order to get further information about their virulence, pathogenicity and survival under stress conditions. Furthermore, these data pave the way for identification of biomarkers to trace sources of contamination with food-borne microorganisms and their endo- and exotoxins in order to ensure food safety and prevent the outbreak of food-borne diseases. Therefore, detection of pathogens and their toxins during production, transport and before consume of food produce, as well as protection against food spoilage is a task of great social, economic and public health importance.

The Peripheral Blood Eosinophil Proteome

A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of platelets that routinely contaminate purified eosinophils and report the variability in the eosinophil proteome among five individuals and proteomic changes accompanying acute activation of eosinophils by interleukin-5. Our deep coverage and quantitative analyses fill an important gap in the existing maps of the human proteome and will enable the strategic use of proteomics to study eosinophils in human diseases.

Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O-formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20(o) C) induces N-formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N-formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below -20(o) C.

Circulating Exosomes in Pancreatic Cancer: Will They Succeed on the Long, Littered Road to Early Detection Marker?

Pancreatic cancer is the fourth leading cause of cancer death in the US and is projected to be the second leading cause of cancer death by 2020. The high mortality of pancreatic ductal adenocarcinoma (PDAC),3 the most common form of pancreatic cancer, is largely a consequence of diagnosis at an advanced stage, when the tumor can no longer be surgically resected for cure. Nevertheless, symptoms rarely develop with early disease, and established risk factors for PDAC—tobacco smoking, obesity, personal history of chronic pancreatitis or diabetes, and family history of pancreatic cancer—are insufficient to risk-stratify the population to facilitate disease screening. Despite extensive research efforts and many potential candidates, no noninvasive biomarker has reached the clinic with utility for early detection of PDAC. Experimental studies indicate that more than a decade elapses from formation of the founder malignant clone to a patient's diagnosis, suggesting a window of opportunity for early detection. With this issue in mind, 2 recent studies have exposed new and interesting biology related to the release of exosomes from pancreatic tumors into peripheral blood (1, 2), raising the possibility of a noninvasive tool for risk stratification and earlier PDAC diagnosis. Exosomes are small, extracellular membrane–enclosed vesicles released from most cell types. Because they initially form from intracellular compartments, they contain nucleic acids and proteins, which can be transferred to other cells upon fusion with their extracellular membrane. Although the full spectrum of exosome biology remains to be defined, it is increasingly clear that exosomes are secreted from cancer cells at higher rates than from healthy cells and are important in facilitating cancer progression and spread (3). By use of proteomics, Melo et al. (1) compared exosomes secreted from cancer cell lines and nontumorigenic cells and identified glypican 1 (GPC1) as a membrane-bound protein preferentially present …