Kelly L. Wormwood

Automated Mass Spectrometry-Based Functional Assay for the Routine Analysis of the Secretome

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.

A Pilot Proteomic Analysis of Salivary Biomarkers in Autism Spectrum Disorder

Autism spectrum disorder (ASD) prevalence is increasing, with current estimates at 1/68–1/50 individuals diagnosed with an ASD. Diagnosis is based on behavioral assessments. Early diagnosis and intervention is known to greatly improve functional outcomes in people with ASD. Diagnosis, treatment monitoring and prognosis of ASD symptoms could be facilitated with biomarkers to complement behavioral assessments. Mass spectrometry (MS) based proteomics may help reveal biomarkers for ASD. In this pilot study, we have analyzed the salivary proteome in individuals with ASD compared to neurotypical control subjects, using MS‐based proteomics. Our goal is to optimize methods for salivary proteomic biomarker discovery and to identify initial putative biomarkers in people with ASDs. The salivary proteome is virtually unstudied in ASD, and saliva could provide an easily accessible biomaterial for analysis. Using nano liquid chromatography‐tandem mass spectrometry, we found statistically significant differences in several salivary proteins, including elevated prolactin‐inducible protein, lactotransferrin, Ig kappa chain C region, Ig gamma‐1 chain C region, Ig lambda‐2 chain C regions, neutrophil elastase, polymeric immunoglobulin receptor and deleted in malignant brain tumors 1. Our results indicate that this is an effective method for identification of salivary protein biomarkers, support the concept that immune system and gastrointestinal disturbances may be present in individuals with ASDs and point toward the need for larger studies in behaviorally‐characterized individuals. Autism Res 2015, 8: 338–350.

Mass spectrometry for the detection of potential psychiatric biomarkers

The search for molecules that can act as potential biomarkers is increasing in the scientific community, including in the field of psychiatry. The field of proteomics is evolving and its indispensability for identifying biomarkers is clear. Among proteomic tools, mass spectrometry is the core technique for qualitative and quantitative identification of protein markers. While significant progress has been made in the understanding of biomarkers for neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis and Parkinson’s disease, psychiatric disorders have not been as extensively investigated. Recent and successful applications of mass spectrometry-based proteomics in fields such as cardiovascular disease, cancer, infectious diseases and neurodegenerative disorders suggest a similar path for psychiatric disorders. In this brief review, we describe mass spectrometry and its use in psychiatric biomarker research and highlight some of the possible challenges of undertaking this type of work. Further, specific examples of candidate biomarkers are highlighted. A short comparison of proteomic with genomic methods for biomarker discovery research is presented. In summary, mass spectrometry-based techniques may greatly facilitate ongoing efforts to understand molecular mechanisms of psychiatric disorders.

The potential of biomarkers in psychiatry: focus on proteomics

Abstract The etiology and pathogenesis of many psychiatric disorders are unclear with many signaling pathways and complex interactions still unknown. Primary information provided from gene expression or brain activity imaging experiments is useful, but can have limitations. There is a current effort focusing on the discovery of diagnostic and prognostic proteomic potential biomarkers for psychiatric disorders. Despite this work, there is still no biological diagnostic test available for any mental disorder. Biomarkers may advance the care of psychiatric illnesses and have great potential to knowledge of psychiatric disorders but several drawbacks must be considered. Here, we describe the potential of proteomic biomarkers for better understanding and diagnosis of psychiatric disorders and current putative biomarkers for schizophrenia, depression, autism spectrum disorder and attention deficit/hyperactivity disorder.

A pilot proteomic study of protein markers in autism spectrum disorder.

Autism spectrum disorder (ASD) diagnosis is increasing, with 1/88 children believed to be affected by the disorder, with a most recent survey suggesting numbers as high as 1/50. Treatment and understanding of ASD causes is a pressing health concern. ASD protein biomarkers may provide clues about ASD cause. Protein biomarkers for ASDs could be used for ASD diagnosis, subtyping, treatment monitoring, and identifying therapeutic targets. Here, we analyzed the sera from seven children with ASD and seven matched controls using Tricine gel electrophoresis (Tricine‐PAGE) and LC‐MS/MS. Overall, we found increased levels of apolipoproteins ApoA1 and ApoA4, involved in cholesterol metabolism and of serum paraoxanase/arylesterase 1, involved in preventing oxidative damage, in the sera of children with ASD, compared with their matched controls. All three proteins are predicted to interact with each other and are parts of high‐density lipoproteins. Further studies are needed to validate these findings in larger subject numbers.

Salivary proteomics and biomarkers in neurology and psychiatry

Biomarkers are greatly needed in the fields of neurology and psychiatry, to provide objective and earlier diagnoses of CNS conditions. Proteomics and other omics MS‐based technologies are tools currently being utilized in much recent CNS research. Saliva is an interesting alternative biomaterial for the proteomic study of CNS disorders, with several advantages. Collection is noninvasive and saliva has many proteins. It is easier to collect than blood and can be collected by professionals without formal medical training. For psychiatric and neurological patients, supplying a saliva sample is less anxiety‐provoking than providing a blood sample, and is less embarrassing than producing a urine specimen. The use of saliva as a biomaterial has been researched for the diagnosis of and greater understanding of several CNS conditions, including neurodegenerative diseases, autism, and depression. Salivary biomarkers could be used to rule out nonpsychiatric conditions that are often mistaken for psychiatric/neurological conditions, such as fibromyalgia, and potentially to assess cognitive ability in individuals with compromised brain function. As MS and omics technology advances, the sensitivity and utility of assessing CNS conditions using distal human biomaterials such as saliva is becoming increasingly possible.

Mass Spectrometry for Proteomics-Based Investigation

Within the past years, we have witnessed a great improvement in mass spectrometry (MS) and proteomics approaches in terms of instrumentation, protein fractionation, and bioinformatics. With the current technology, protein identification alone is no longer sufficient. Both scientists and clinicians want not only to identify proteins but also to identify the proteins posttranslational modifications (PTMs), protein isoforms, protein truncation, protein-protein interaction (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics and strategies to identify proteins, proteins PTMs, protein isoforms, protein truncation, PPIs, and protein quantitation. We also discuss the strengths and weaknesses within this field. Finally, in our concluding remarks we assess the role of mass spectrometry and proteomics in scientific and clinical settings in the near future. This chapter provides an introduction and overview for subsequent chapters that will discuss specific MS proteomic methodologies and their application to specific medical conditions. Other chapters will also touch upon areas that expand beyond proteomics, such as lipidomics and metabolomics.

Comparative two‐dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two‐dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P‐value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC‐MS/MS. Alpha‐amylase, CREB‐binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down‐regulated in ASD. Increased expression of proto‐oncogene Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin‐inducible protein precursor, Mucin‐16, Ca binding protein migration inhibitory factor‐related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.

Autism spectrum disorder: An omics perspective

Current directions in autism spectrum disorder (ASD) research may require moving beyond genetic analysis alone, based on the complexity of the disorder, heterogeneity and convergence of genetic alterations at the cellular/functional level. Mass spectrometry (MS) has been increasingly used to study CNS disorders, including ASDs. Proteomic research using MS is directed at understanding endogenous protein changes that occur in ASD. This review focuses on how MS has been used to study ASDs, with particular focus on proteomic analysis. Other neurodevelopmental disorders have been investigated using MS, including fragile X syndrome (FXS) and Smith‐Lemli‐Opitz Syndrome (SLOS), genetic syndromes highly associated with ASD comorbidity.

Proteomics and Cholesterol in Autism

Autism Spectrum Disorder (ASD) diagnosis is increasing worldwide. ASDs are characterized by impaired social function, stereotyped behaviors/interests and communication deficits. ASD causes are poorly understood and treatments are largely limited to behavioral interventions once problems have developed and been detected. Here we discuss the potential use of mass spectrometry and proteomics in early diagnosis of ASD. The potential link between at least some subtypes of ASD, the cholesterol system and proteins that interact with cholesterol is also discussed.