A Proteomics Investigation of Salivary Profiles as Potential Biomarkers for Autism Spectrum Disorder (ASD).

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that affects approximately 1/68 children, with a more recent study suggesting numbers as high as 1/36. According to Diagnostic and Statistical Manual of Mental Disorders, the etiology of ASD is unknown and diagnosis of this disorder is behavioral. There is currently no biomarker signature for ASD, however, identifying a biomarker signature is crucial as it would aid in diagnosis, identifying treatment targets, monitoring treatments, and identifying the etiology of the disorder. Here we used nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the saliva from individuals with ASD and matched controls in a 14 vs 14 study. We found numerous proteins to have statistically significant dysregulations, including lactotransferrin, transferrin, polymeric immunoglobulin receptor, Ig A L, Ig J chain, mucin 5 AC, and lipocalin 1 isoform X1. These findings are consistent with previous studies by our lab, and others, and point to dysregulations in the immune system, lipid metabolism and/or transport, and gastrointestinal disturbances, which are common and reoccurring topics in ASD research.

Mass Spectrometry for Proteomics-Based Investigation.

Within the past years, we have witnessed a great improvement is 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 the proteins, but also to identify the protein's post-translational modifications (PTMs), protein isoforms, protein truncation, protein-protein interactions (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics, and strategies to identify proteins, protein's 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 the 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.

Proteomics and Non-proteomics Approaches to Study Stable and Transient Protein-Protein Interactions.

Of the 25,000-30,000 human genes, about 2 % code for proteins. However, there are about 1-2 million protein entities. This is primarily due to alternative splicing, post-translational modifications (PTMs) or protein-protein interactions. Proteomics sets out to identify proteins, their sequence and known modifications as well as their quantitation in a biological sample for the purpose of understanding biological processes, protein cellular functions, and their physiological and pathological involvement in diseases.Proteins interact at the molecular level with other proteins, nucleic acids, lipids, carbohydrates and metabolites to perform numerous cellular activities. Protein complexes can consist of sets of more stably (stable PPIs) and less stably (transient PPIs) interacting proteins or combination of both. Here, we discuss the proteomics and non-proteomics approaches to study stable and transient PPIs.

Identification of Posttranslational Modifications (PTMs) of Proteins by Mass Spectrometry.

There are only 30,000 human genes, which, according to the central dogma from biology, it means that there should be 30,000 mRNA and 30,000 proteins. However, there are at least 1-2 million protein entities that are expressed in a cell at a given time. This is primarily due to alternative splicing in different cells and tissues, which may lead to expression of different protein isoforms within one cell, but also different protein isoforms in different tissues. A new level of complexity of proteins and protein isoforms is then given by posttranslational modifications (PTMs) of proteins. Here, we discuss the PTMs in proteins and how they are identified by mass spectrometry and proteomics, with specific examples on identification of acetylation, phosphorylation, glycosylation, alkylation, hydroxinonenal-modification or assignment of intramolecular and intermolecular disulfide bridges.

Mass Spectrometry- and Computational Structural Biology-Based Investigation of Proteins and Peptides.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction, and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation, we will describe two proteins-lysozyme and myoglobin. As an application of MS-based assignment of disulfide bridges, the case of the spider venom polypeptide Phα1ß will also be discussed.

Role of Mass Spectrometry in Investigating a Novel Protein: The Example of Tumor Differentiation Factor (TDF).

Better understanding of central nervous system (CNS) molecules can include the identification of new molecules and their receptor systems. Discovery of novel proteins and elucidation of receptor targets can be accomplished using mass spectrometry (MS). We describe a case study of such a molecule, which our lab has studied using MS in combination with other protein identification techniques, such as immunohistochemistry and Western Blotting. This molecule is known as tumor differentiation factor (TDF), a recently-found protein secreted by the pituitary into the blood. TDF mRNA has been detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. Currently TDF has an unclear function, and prior to our studies, its localization was only minimally understood, with no understanding of receptor targets. We investigated the distribution of TDF in the rat brain using immunohistochemistry (IHC) and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions, in specific neurons but not astrocytes. We found TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized to neurons, not to astrocytes. Our group also conducted studies to identify the TDF receptor (TDF-R). Using LC-MS/MS and Western blotting, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) as potential TDF-R candidates in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. These studies have combined directed protein identification techniques with mass spectrometry to increase our understanding of a novel protein that may have distinct actions as a hormone in the body and as a growth factor in the brain.

Mass Spectrometry for the Study of Autism and Neurodevelopmental Disorders.

Mass spectrometry (MS) has been increasingly used to study central nervous system (CNS) disorders, including autism spectrum disorders (ASDs). The first studies of ASD using MS focused on the identification of external toxins, but current research is more directed at understanding endogenous protein changes that occur in ASD (ASD proteomics). This chapter focuses on how MS has been used to study ASDs, with particular focus on proteomic analysis. Other neurodevelopmental disorders have been investigated using this technique, including genetic syndromes associated with autism such as fragile X syndrome (FXS) and Smith-Lemli-Opitz Syndrome (SLOS).

Protein Biomarkers in Major Depressive Disorder: An Update.

Major depressive disorder (MDD) is common. Despite numerous available treatments, many individuals fail to improve clinically. Diagnosis of MDD continues to be commonly accomplished via behavioral rather than biological methods. Biomarkers may provide objective diagnosis of MDD, and could include measurements of genes, proteins, and patterns of brain activity. Proteomic analysis and validation of biomarkers is less explored than other areas of biomarker research in MDD. Mass spectrometry (MS) is a comprehensive, unbiased means of proteomic analysis, which can be complemented by directed protein measurements, such as Western Blotting. Prior studies have focused on MS analysis of several human biomaterials in MDD, including human post-mortem brain, cerebrospinal fluid (CSF), blood components, and urine. Further studies utilizing MS and proteomic analysis in MDD may help solidify and establish biomarkers for use in diagnosis, identification of new treatment targets, and understanding of the disorder. A biomarker or a biomarker signature that facilitates a convenient and inexpensive predictive test for depression treatment response is highly desirable.

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.

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.

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.

The potential of biomarkers in psychiatry: focus on proteomics.

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.

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.

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 protein's 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, protein's 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.

Utility of computational structural biology in mass spectrometry.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation we will describe two proteins, lysozyme and myoglobin.

Mass spectrometric analysis of post-translational modifications (PTMs) and protein-protein interactions (PPIs).

Of the 25,000-30,000 human genes, about 2 % code for proteins. However, there are about one to two million protein entities. This is primarily due to alternative splicing and post-translational modifications (PTMs). Identifying all these modifications in one proteome at a particular time point during development or during the transition from normal to cancerous cells is a great challenge to scientists. In addition, identifying the biological significance of all these modifications, as well as their nature, such as stable versus transient modifications, is an even more challenging. Furthermore, interaction of proteins and protein isoforms that have one or more stable or transient PTMs with other proteins and protein isoforms makes the study of proteins daunting and complex. Here we review some of the strategies to study proteins, protein isoforms, protein PTMs, and protein-protein interactions (PPIs). Our goal is to provide a thorough understanding of these proteins and their isoforms, PTMs and PPIs and to shed light on the biological significance of these factors.

Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF).

Better understanding of central nervous system (CNS) molecules can include the identification of new molecules and their receptor systems. Discovery of novel proteins and elucidation of receptor targets can be accomplished using mass spectrometry (MS). We describe a case study of such a molecule, which our lab has studied using MS in combination with other protein identification techniques, such as immunohistochemistry (IHC) and Western blotting. This molecule is known as tumor differentiation factor (TDF), a recently-found protein secreted by the pituitary into the blood. TDF mRNA has been detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. Currently TDF has an unclear function, and prior to our studies, its localization was only minimally understood, with no understanding of receptor targets. We investigated the distribution of TDF in the rat brain using IHC and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions, in specific neurons but not astrocytes. We found TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized to neurons, not to astrocytes. Our group also conducted studies to identify the TDF receptor (TDF-R). Using LC-MS/MS and Western blotting, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) as potential TDF-R candidates in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cell fibroblasts or fibroblast-like cells. These studies have combined directed protein identification techniques with mass spectrometry to increase our understanding of a novel protein that may have distinct actions as a hormone in the body and as a growth factor in the brain.

Mass spectrometry for the study of autism and neurodevelopmental disorders.

Mass spectrometry (MS) has been increasingly used to study central nervous system disorders, including autism spectrum disorders (ASDs). The first studies of ASD using MS focused on the identification of external toxins, but current research is more directed at understanding endogenous protein changes that occur in ASD (ASD proteomics). This chapter focuses on how MS has been used to study ASDs, with particular focus on proteomic analysis. Other neurodevelopmental disorders have been investigated using this technique, including genetic syndromes associated with autism such as fragile X syndrome and Smith-Lemli-Opitz syndrome.

Biomarkers in major depressive disorder: the role of mass spectrometry.

Major depressive disorder (MDD) is common. Despite numerous available treatments, many individuals fail to improve clinically. MDD continues to be diagnosed exclusively via behavioral rather than biological methods. Biomarkers-which include measurements of genes, proteins, and patterns of brain activity-may provide an important objective tool for the diagnosis of MDD or in the rational selection of treatments. Proteomic analysis and validation of its results as biomarkers is less explored than other areas of biomarker research in MDD. Mass spectrometry (MS) is a comprehensive, unbiased means of proteomic analysis, which can be complemented by directed protein measurements, such as Western Blotting. Prior studies have focused on MS analysis of several human biomaterials in MDD, including human post-mortem brain, cerebrospinal fluid (CSF), blood components, and urine. Further studies utilizing MS and proteomic analysis in MDD may help solidify and establish biomarkers for use in diagnosis, identification of new treatment targets, and understanding of the disorder. The ultimate goal is the validation of a biomarker or a biomarker signature that facilitates a convenient and inexpensive predictive test for depression treatment response and helps clinicians in the rational selection of next-step treatments.