Mass Spec Flashcards
coimmunoprecipitation determines whether two or more KNOWN proteins interact with each other–see if prot X interacts with Y–>immunoppt X and test for presence of Y in the ppt
ya
what is the upper limit for MS?
500 000 Da
advantage of MS?
can use only picomolars of amounts of prots; possess accuracy of ~0.01% for 25 kDa protein–>(25 000+/- 2.5) Da
where does Trpsin cleave?
K and R, but not before P (proline)– won’t cleave in front of P?
MS more sensitive–>has a 0.001 Da error
ya
can’t tell apart Leu and Isoleu bc they have the same MW
ya
MS tells us the proteins present but not how they interact with each other–bridged or direct
ya
what are the targets for chemical crosslinking?
primary amines (NH2)
carboxyls (COOH)
sylfhydryls (SH)
carbonyls (-CO-)
what are the physical and chemical properties to look at when choosing a cross-linker?
chemical specificity; spacer arm length/cleavibility; solubility and permeability; reversible/irreversible crosslinker
DSG, disuccinymidyl glutarate is a common crosslinker-symmetrical, has two reactive groups
ya
one problem with LC-MS is identifying which proteins interact nonspecifically with the solid-phase, affinity reagent, or tag–>challenging task
ya
what is bimolecular fluorescence complimentation?
GFP links to N and G–>when not combined, so fluorscence; when combined–>fluorescence–>shows if interaction is happening or not
–good if you can’t isolate proteins physically
why use library-based methods?
biological responses often very complex and highly variable
protein-protein interactions are weak and transient in nature–>sometimes cannot physically capture
limitations of yeast-2-hybrid?
high false pos and neg rates
requires overexpressed fusion proteins localized to yeast nucleus:
1. overexpression, nonspecific interactions
2. observed interactions may never occur in vivo
3. mammalian proteins not always properly PTM’d
biased against certain classes of proteins
e.g. transcription factors (other DNA binding proteins); integral membrane proteins
