proteomics Flashcards
proteomics
study of proteins, how they’re modified, their expression and interactions
expression proteomics
identifying levels of proteins - how much given protein expressed or not in cell type
quantifying proteome in 2 or more states e.g. disease. vs normal
functional proteomics
identifying protein-protein interactions
quantifying these interactions to determine protein function
how proteins interact to derive certain function
the proteome
all different proteins forms expressed from genome
how can proteins be extracted
detergent e.g. SDS (to break open lipid bilayer)
homogenise
sonicate
fractionate cells
to look at specific portion of cell contents
break up cell and then isolate organelles from cytoplasm through centrifugation
e.g. if want to focus on nucleus
SDS-PAGE
PAGE = polyacrylamide gel electrophoresis
SDS unfolds protein and coats with negative charge
proteins own charge then irrelevant
SDS-PAGE separates on basis of size
stain to see result e.g.
coomassie brillant blue
silver
2D gels
separate on basis charge and size
run SDS-PAGE on a single lane. then run sideways without SDS
can screen drugs with this - to see changes to proteins due to drugs
high pressure liquid chromatography (HPLC)
HPLC metal tube under pressure
sample injecter, pump, solvent reservoir that lets buffer flow through tube
proteins flow with buffer and are separated in some way e.g size, charge, shape, hydrophobic-ness, phosphorylation
HPLC clinical application
used to screen newborns for sickle cell
matrix-assisted laser depolarisation/ionisation - time of flight mass spectrometry
sample added to plate within a matrix and hit with laser which vaporises it –> charged ions accelerated by electrical gradient and timed (large ones hit detector last)
size and charge
liquid chromatography coupled with tandem mass spectrometry
liquid chromatography separates proteins based on characteristic, e.g. size, and 2 rounds mass spec to identify separated proteins
sample hit w laser and subject to MS to select an ion (based on characteristic. ion passed through collision cell which fragments it into smaller pieces. fragmented ions then analysed with another round MS , to obtain reading of spectrum
