Glycans and Mass Spectrometry Flashcards
1
Q
Influenza Viruses
A
- RNA viruses
- 4 types A-D
- key surface proteins are Neuraminidase monomer (forms homotetramer on virus surface( (hydrolytic enzyme) and Hemagglutinin trimer (lectin)
- different serotypes of these are different structural variants
- 18 serotypes of HA and 11 serotypes of NA
- pathogens use sugar recognition phenomena to initiate infection
2
Q
Influenza Infection
A
- attachment: recognises sugar sequences to initiate infection (sialic acid capping group recognises glycans)
- endocytosis
- replication
- budding
- release: NA cleaves sialic acid residues stopping new particles getting stuck on host cell surface
- NA allows movement of viral particles through mucinous layer to underlying cell surface
3
Q
Haemagglutinin receptors
A
- human receptor: a-2-6 glycosidic bond to sialic acid
- avian receptor: a-2-3 glycosidic bond to sialic acid
- mutations of HA switch species infectivity and change the sugar recognition properties
4
Q
H1N1 Pandemic
A
- mixing of a2-3 and 2-6 in swine species at the same time led to reassortment of the virus
- new strain caused 20-27% of world population to be infected
5
Q
Neuraminidase Inhibitors
A
- tamiflu and relenza both bind sialic acid
- HA (lectin) and NA (enzyme) bind sialic acid
- carb recognition by HA uses 3 shallow CRD : weak affinity binding : bad drug target
- NA uses a high strength deep binding pocket therefore is a strong drug target
6
Q
Drug Discovery
A
- soaked fragments with NA to visualize key interactions and residues in the pocket (from sialic acid in the active site of enzyme and the aa making up deep pocket of enzymes)
- found lots of charge charge interactions and H bonding
7
Q
Tamiflu
A
- NA inhibitor
- changed structure of glycerol side chain compared to sialic acid (hydroxyls needed in key interactions with Glu in enzyme active site)
- by putting an ethyl group here the H bonding ability is lost
- crystal structure showed the binding is affected (Glu shifts orientation and interacts with another Arg residue
- by inhibiting NA there is no virion release from the cell
8
Q
Glycomics
A
- determining the glycan repertoire in the cell, tissues, organs, etc to define their function
- need prior knowledge of glycan biosynthetic pathways
9
Q
Glycomics Screening : Glycoproteins
A
- carboxymethylation, proteolysis, C18 chromatography
- reductive elimination and analysis (remove N linked glycans and removes O glycans)
- permethylation and chromatography for MS analysis
10
Q
Glycomics Screening : Glycolipids
A
- chloroform, methanol, water extractions (remove glycolipid derived glycans)
- digestion, extraction
- permethylation and chromatography
- MS analysis
11
Q
Permethylation
A
- chemical derivitisation of sugars
- replace hydrophilic side chains with -omethyl
- MS works better with hydrophobic molecules
- just increases signal:noise to improve quality and intensity of data
rmethylation consists of replacing all hydrogens attached to oxygen and nitrogen atoms with methyl groups and is a necessary step in the structural characterization of glycans by mass spectrometry (MS). Currently, only permethylation can enable MS to provide sequence, branching, and linkage information for glycans. (3) Unfortunately, permethylation is a difficult and time-consuming process that is not easily adapted to large numbers of samples and requires specialized training.
12
Q
Glycan Molecular Ions
A
- usually contains M + Na+ molecular ions
- reducing end : OCH3 (31) and Na (23)
- nonreducing end : CH3 (15)
- need to take account of these when calculating glycan sequence from spectra
- MI gives charge to see the M/Z
13
Q
MALDI Mapping
A
- structural isomers cannot be differentiated via MS
- knowledge of the biosynthetic pathways helps our identification (only 1 structure will be allowed biosynthetically)
- use the masses to work out composition of sugars and use knowledge of pathways to identify structure
14
Q
Glycan Spectra Characteristics
A
- high mannose : low mass
- complex : high mass
- see notes and memorise numbers *
1579 : first high mannose glycan
15
Q
MALDI Mapping and Disease
A
- altered N-glycome used to diagnose early stage liver cancer
- bisected structures are indicative of liver disease
