Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques

Identification of proteins in the postsynaptic density fraction by mass spectrometry

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates. Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95. The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms

Quantitative analysis of PPT1 interactome in human neuroblastoma cells

Peer reviewe

System level dynamics of post-translational modifications

Attempts to characterize cellular behaviors with static, univariate measurements cannot fully capture biological complexity and lead to an inadequate interpretation of cellular processes. Significant biological insight can be gleaned by considering the contribution of dynamic protein post-translational modifications (PTMs) utilizing systems-level quantitative analysis. High-resolution mass spectrometry coupled with computational modeling of dynamic signal–response relationships is a powerful tool to reveal PTM-mediated regulatory networks. Recent advances using this approach have defined network kinetics of growth factor signaling pathways, identified systems level responses to cytotoxic perturbations, elucidated kinase–substrate relationships, and unraveled the dynamics of PTM cross-talk. Innovations in multiplex measurement capacity, PTM annotation accuracy, and computational integration of datasets promise enhanced resolution of dynamic PTM networks and further insight into biological intricacies

Development of novel proteomic strategies to dissect plant phosphoproteomic signaling under environmental stresses

Protein phosphorylation is one of the important signaling mechanisms in plants which transduces environmental stimuli such as salinity, microbes, and hormones into intracellular signals and activates plant defense mechanisms. Thus, understanding the correlation between environmental stresses and alteration of plant phosphorylation requires system-wide phosphoproteomic analysis, which includes identification of kinase-substrate complexes and measurement of phosphorylation-mediated signaling changes. However, identification and quantification of plant phosphoproteome remains challenging due to the highly dynamic nature of plant proteome, interferences of cell wall, pigments, and secondary metabolites. Recently, mass spectrometry (MS) has been integrated with phosphopeptide enrichment approaches for identifying thousands of phosphorylation sites and for quantifying phosphoprotein stoichiometry. Although MS-based phosphoproteomics has revealed the global phosphorylation changes related to different physiological states of plants, many kinase-substrate networks involved in essential signaling pathways, such as the ABA-induced SNF-1-related protein kinase 2 (SnRK2) pathway and the mitogen-activated protein kinases (MAPKs) cascades, are still not completely understood. This dissertation discusses strategies for improving plant sample preparation and for identifying the direct substrates of the plant kinases. Chapter one highlights the low phosphopeptide identification rate by mass spectrometry. Chapter two details the development of a sample preparation protocol for the plant phosphoproteome analysis, and the application of the protocol for the study of tomato cold-induced phosphoproteomic changes. Chapter three shows the development of a novel approach for identification of the direct substrates of the plant kinases, whose activation regulates the signaling transductions of plant stress defense mechanisms

Proteome and phospho-proteome study of Molm-13 cell line in Acute Myeloid Leukemia (AML)

Curs 2015-2016The main goal of this thesis was to study the changes in the proteome of Molm-13 cells from Acute Myeloid Leukemia when treated with a proliferation and cell differentiation inductor (FLT3 ligand) and with an apoptosis inductor (Okadaic Acid, OA, FLT3 ligand binds to FLT3 receptor which auto phosphorylates inducing cell proliferation and differentiation. FLT3 receptor is commonly mutated in AML, this mutation is named FLT3-ITD. Signaling pathways like RAS/MAP kinase, AKT/PI-3 kinase and JAK/ STAT are activated depending on ITD localization. Some other cells were treated with Okadaic Acid, a toxin and a potent inhibitor of the Serine/ Threonine (Ser/Thr) protein phosphatases 1 and 2, which blocks the activation of ERK5 a protein present in the MAP kinase pathway. To perform proteomics analysis, protein content of each sample was digested by Filter Aided Sample Preparation Double Digestion method and a peptide aliquot was enriched by Titanium Dioxide to get phosphorylated peptides. Samples were analysed by mass spectrometry with the Orbitrap Elite. More than 3000 proteins were identified in the different samples analysed. Bcl-6, Bcl-2, STAT and TNFAIP8 are important proteins which contribute to tumor cell progression and inhibit the apoptosis process by inhibiting caspases function. These mentioned proteins were found in Wild type (untreated cells) and FLT3- ligand induced cells. Whereas in Okadaic Acid treated cells different proteins were expressed as apoptotic inducer, these proteins are p53 and TNF along more proteins. FLT3- ITD mutation is probably localized in the Endoplasmatic Reticulum (ER)

Development of sensitive proteomic approaches for protein tyrosine phosphorylation detection.

The elucidation of the complex array of cell signalling cascades is imperative for a deeper understanding of cell biology in both physiological and patho-physiological states. Extensive biochemical characterisation of signalling networks has revealed the importance of post-translational modifications (PTMs), particularly phosphorylation. Signalling via protein phosphorylation occurs across homeostatic proliferative, differentiative and anti-apoptotic events. Dysregulation of the kinase signalling pathways as well as mutations in kinases involved in phosphorylation have been implicated in a number of pathologies such as cancer or immune deficiencies. While it is estimated that 50% of all proteins are phosphorylated during their lifetime, phosphorylated proteins are present in relatively low abundance compared to their non-phosphorylated counterparts. The rarity of phosphorylation, which occurs on serine, threonine and tyrosine residues, has prompted the development of sensitive approaches to improve phosphorylation characterisation. Proteomic-based strategies offer novel approaches to overcome the limitations of currently available strategies for phosphoprotein analysis. The research presented within describes the development of proteomic-based methodologies for phosphotyrosine identification, quantitation and characterisation. These methods utilise the antiphosphotyrosine. Antibody 4G10 along with other MS-compatible approaches for phosphotyrosine enrichment prior to MS analysis. Methods for more targeted phosphoprotein analyses involved coupling of 4G10 covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. These 4G10-coupled beads successfully enriched tyrosine phosphopeptides derived from tryptic phosphoprotein digests for identification and characterisation of phosphopeptides using MALDI-TOF/TOF MS analysis. The limited capacity of the magnetic bead approach for analysis of more complex samples necessetated the development of a more global proteomic strategy for tyrosine phosphorylation analysis. A global strategy that provides not only qualitative pTyr information but also shows quantitative changes that occur with pTyr signalling is imperative for detailed signalling cascade analyses. The global approach presented here utilised the 4G10 Ab/bead approach as well as Hydrophilic interaction chromatography (HILIC) for the enrichment of pTyr peptides from complex samples isotopically-labelled to quantify tyrosine phosphorylation after LC-MALDI-TOF/TOF MS analysis. Aspects of this approach were modified to improve phosphopeptide detection and characterisation, including the development of a novel optimised matrix-deposition strategy for LCMALDI-TOF/TOF MS. The strategy, termed EZYprep LC, allowed the effective use of the atypical 2,5-DHB matrix with phosphoric acid to improve phosphopeptide ionisation and subsequently identify and characterise more phosphorylation sites on phosphoprotein samples compared with LC-ESI-IT-MS/MS. Another aspect of the global strategy was the development of a modified isotope protein coded label strategy (modified ICPL). The optimised ICPL approach ensured quantitative information from a larger sub-set of peptides after tryptic digest of complex samples. The improved ability to quantify using this approach was highlighted by a comparative analysis of complex cell lysates labelled using the conventional ICPL strategy and the modified ICPL strategy. The modified ICPL labelling strategy identified more proteins and provided more quantitative information that the conventional ICPL methodology. As such, the global phospho-tyrosine strategy, combined the modified ICPL labelling and 4G10 Ab/bead enrichment with peptide fractionation and MALDI-TOF/TOF MS analysis, was subsequently utilised to identify and quantify tyrosine phosphorylation occurring in insulin-stimulated insulin receptor A- and B-subtypes.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201