Roshanak Aslebagh

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.

Using Breast Milk to Assess Breast Cancer Risk: The Role of Mass Spectrometry-Based Proteomics

Although mammography and treatment advances have led to declines in breast cancer mortality in the United States, breast cancer remains a major cause of morbidity and mortality. Breast cancer in young women is associated with increased mortality and current methods of detecting breast cancers in this group of women have known limitations. Tools for accurately assessing personal breast cancer risk in young women are needed to identify those women who would benefit the most from earlier intervention. Proteomic analysis of breast milk could identify biomarkers of breast cancer risk and provide a tool for identifying women at increased risk. A preliminary analysis of milk from four women provides a proof of concept for using breast milk to assess breast cancer risk.

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-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins.

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.

Advances in Mass Spectrometry for Glycoscreening and Sequencing in Biomedical Research

During the past several years, applications of Mass Spectrometry (MS) in the biomedical research increased considerably. While MS is for many years heavily used in proteomics, for protein identification and quantification as well as for biomarker discovery, for a long time its applications in glycomics were limited, mainly because of the challenging conditions required for the ionization and detection of most carbohydrate classes. However, due to the development of high performance analytical instrumentation, MS in particular with Electrospray (ESI) and Matrix Assisted Laser Desorption/Ionization (MALDI) started to be intensively applied also to the analysis of post-translational modifications such as glycosylation, acetylation or phosphorylation. Focus on MS-based glycosylation is, however, scarce. Therefore, analysis of glycoproteins in particular diseases through glycoscreening and sequencing is another new MS-based avenue, yet to be pursued. In this context, we discuss briefly here the recent advances of MS in glycomics and glycoscreening and their applications in biomedical research, with a particular emphasis on cancer, lysosomal storage and bacterial diseases.

Proteomics analysis of human breast milk to assess breast cancer risk

Detection of breast cancer (BC) in young women is challenging because mammography, the most common tool for detecting BC, is not effective on the dense breast tissue characteristic of young women. In addition to the limited means for detecting their BC, young women face a transient increased risk of pregnancy‐associated BC. As a consequence, reproductively active women could benefit significantly from a tool that provides them with accurate risk assessment and early detection of BC. One potential method for detection of BC is biochemical monitoring of proteins and other molecules in bodily fluids such as serum, nipple aspirate, ductal lavage, tear, urine, saliva and breast milk. Of all these fluids, only breast milk provides access to a large volume of breast tissue, in the form of exfoliated epithelial cells, and to the local breast environment, in the form of molecules in the milk. Thus, analysis of breast milk is a non‐invasive method with significant potential for assessing BC risk. Here we analyzed human breast milk by mass spectrometry (MS)‐based proteomics to build a biomarker signature for early detection of BC. Ten milk samples from eight women provided five paired‐groups (cancer versus control) for analysis of dysregulatedproteins: two within woman comparisons (milk from a diseased breast versus a healthy breast of the same woman) and three across women comparisons (milk from a woman with cancer versus a woman without cancer). Despite a wide range in the time between milk donation and cancer diagnosis (cancer diagnosis occurred from 1 month before to 24 months after milk donation), the levels of some proteins differed significantly between cancer and control in several of the five comparison groups. These pilot data are supportive of the idea that molecular analysis of breast milk will identify proteins informative for early detection and accurate assessment of BC risk, and warrant further research. Data are available via ProteomeXchange with identifier PXD007066.

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide with up to 80% of patients remaining undiagnosed. OSA has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. We have implemented a rat model of OSA involving the surgical implantation of a tracheal obstructive device. Rats were divided into severe and moderate apnea groups, receiving 23 seconds (severe) or 13 seconds (moderate) apneas per minute, 60 apneas per minute for 8 hours a day over 2 weeks. We recently performed a pilot study using onedimensional polyacrylamide gel electrophoresis (1D PAGE) and nanoliquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) to investigate the protein dysregulations in rat atria which was induced with OSA using the rat model we developed. We found, among others, that some aerobic and anaerobic glycolytic enzymes and Krebs cycle enzymes were downregulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents. Here, we used twodimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoLC-MS/MS as a complementary approach to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We not only found that the entire glycolytic pathway and Krebs cycle are downregulated, but also found evidence that additional enzymes involved in the beta-oxidation, electron transport chain and Krebs cycle anaplerotic reactions were also downregulated. Other protein dysregulations identified are involved in metabolic, structural, or inflammatory pathways, suggesting that these proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia.

Comparative two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of human milk to identify dysregulated proteins in breast cancer

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC‐associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one‐dimensional polyacrylamide gel electrophoresis (1D‐PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC‐MS/MS) analysis. The upregulated proteins in BC versus control are alpha‐amylase, gelsolin isoform a precursor, alpha‐2‐glycoprotein 1 zinc isoform CRA_b partial, apoptosis‐inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.

REVIEW ARTICLES 29 Molecular Life │ VOProteomics of the post-translational modifi cations: the knowns and the unknowns

Abbreviations: PTMs=post-translational modifi cations; PPIs=protein-protein interactions; SDS-PAGE= sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2D-PAGE=two-dimensional polyacrylamide gel electrophoresis; MS=mass spectrometry; ESI=electrospray ionization; MALDI=Matrix Assisted Laser Desorption Ionization; ESI-MS=electrospray ionization mass spectrometry; MALDI-MS=MALDI mass spectrometry; LC-MS/MS=liquid chromato graphy tandem mass spectrometry; ESI-MS/MS=electro spray ionization tandem mass spectrometry; m/z=mass/charge; PNGaseF=peptide-N-glycosidase F; HNE=4-hydroxy-2-nonenal; IMAC=immobilized metal affi nity chromatography; TiMAC=combination of TiO2 and IMAC; MRM=multiple reaction monitoring; DDA=data dependent acquisition; PID=product ion discovery; BSA=bovine serum albumin; DTT=dithiothreitrol; IAA=iodoacetamide.