Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides.

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso-forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein-related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry-based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase.

Children with neurofibromatosis type 1 (NF1) are increasingly recognized as having a high prevalence of social difficulties and autism spectrum disorders (ASDs). We demonstrated a selective social learning deficit in mice with deletion of a single Nf1 allele (Nf1(+/-)), along with greater activation of the mitogen-activated protein kinase pathway in neurons from the amygdala and frontal cortex, structures that are relevant to social behaviors. The Nf1(+/-) mice showed aberrant amygdala glutamate and GABA neurotransmission, deficits in long-term potentiation and specific disruptions in the expression of two proteins that are associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (Adam22) and heat shock protein 70 (Hsp70), respectively. All of these amygdala disruptions were normalized by the additional deletion of the p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1(+/-) mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide insights and therapeutic targets for patients with NF1 and ASDs.

Fish consumption, low-level mercury, lipids, and inflammatory markers in children.

There is considerable evidence that consuming fish has numerous health benefits, including a reduced risk of cardiovascular disease. However, fish is also the primary source of human exposure to mercury (Hg). In a cross-sectional study of 9-11 year old children (N=100), we measured fish consumption, blood lipids, total blood Hg, diurnal salivary cortisol (4 samples collected throughout the day), and performed a proteomic analysis of serum proteins using spectral count shotgun proteomics. Children who consumed fish had a significantly more atheroprotective lipid profile but higher levels of blood Hg relative to children that did not consume fish. Although the levels of blood Hg were very low in these children (M=0.77 µg/L; all but 1 participant had levels below 3.27 µg/L), increasing blood Hg was significantly associated with blunted diurnal cortisol levels. Blood Hg was also significantly associated with acute-phase proteins suggesting systemic inflammation, and several of these proteins were found to significantly reduce the association between Hg and diminished cortisol when included in the model. This study of a pediatric population is the first to document an association between blood Hg, systemic inflammation, and endocrine disruption in humans. Without a better understanding of the long-term consequences of an atheroprotective lipid profile relative to blunted diurnal cortisol and systemic inflammation, a determination of the risk-benefit ratio for fish consumption by children is not possible.

A proteomic survey of rat cerebral cortical synaptosomes.

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.