Mass Spec 2 Flashcards
What are the two ways to identify proteins
Which is better
Peptide mass fingerprinting
Shotgun sequencing (better)
What is the mass redundancy problem
The m/z ratio of a peptide isnt always enough to identify the protein (which is why PMF isn’t as good)
Different sequences could give the same peptide mass
What is tandem mass spectrometry (MS/MS)
Basically doing the mass spec experiment twice
First ionize the sample using either MALDI or Electrospray (ESI)
The first mass spec selects a precursor ion
That precursor ion gets fragments with an inert gas (he or ar) by collision induced dissociation (CID)
Then those fragments of the precursor ion go into another mass spec to be separated and detected
In tandem mass spec what is the shotgun
Shotgun is the thing that breaks the sample into fragments
So the shotgun is the inert gas he or ar
How do you analyze the fragmentation of the peptide in MS/MS
The abc is coming from the amino term end
The xyz is coming from the carboxy term end
So if you have a1 and x3 the subscripts have to add up to the total amount of amino acid residues (4)
How do you get AX
BY
CZ
From the MS/MS fragment
If fragmented in the c aplha and the c=o
In the c=o and N-H
In the N-H and C alpha
Is MS/MS what fragments do we see
The one that keeps the positive charge that we selected it to have
Even though many fragments are made in MSMS which do we focus on
The B and Y ions (fragmented between the c=o and N-H
How do you find the mass of the B type ion and the y type
The B type is the sum of the mass of both residues + 1 (due to the charge)
Y type is same but add the mass of water
How can you check for error when finding the mass of the y and B type ion
The sums of their masses should equal the total mass of the peptide + 2 (because 2 protons)
What residues are hard to distinguish if we are checking their m/z ratio
Leucine and isoleucine because isomers
How do you analyze a mama spectrum
If you subtract the y type ions from each other the difference in mass is the monoisotopic mass of the next amino acid in the sequence
Is using the y type, you get the sequence backwards
If I’m MSMS you see a big leak with a 2+ charge what is it
How do you find the mass
How do you check that it’s the mass of the peptide
It’s the parent ion
Multiple the mass it’s written as by 2 since it’s actually m/2z
This is the M + 2 of the equation so subtract 2 to get the M
can check by adding the the B and y type ions that sum to the total number of amino acids ex. 12aa, sun up b3 and y9)
What is the sequence tag approach of MSMS
You have the entire mass of the peptide and I little bit of sequence info
You don’t have the full sequence
What is the key thing that allows MSMS to work
The orthagonality of the analysis
Which is Being able to use many unrelated ways of analysis together to get a singular solution
Ex. just using the mass of the peptide , or just the short sequence won’t help but together they work
What are the applications of mass spec
Postranslational modification
Deconvoluted soectra
Structural biology
What is a deconvoluted spectrum
Instead of showing us a spectrum of all this different fragments and their peaks
Show a spectrum of the mass of the parent protein using the original spectra
What is RNAse B
The glycosylated form of RNase A
How can a mass spec show us the glycosylation of a protein
What is special about it
Its would have a different mass depending on the amount of sugars added to it
At some point the sugars can be added at different positions but give the same mass, so we can’t tel which position that sugar was added onto
How can you tell if something was shotgun sequenced (MSMS)
There are B and Y type ions in the spectrum
How can shotgun sequencing show is phosphorylation of a protien
There are B type ions that are the same but have the mass difference of a H3PO4
Also there are two peaks for the parent ion , this diff in this is half the mass of the H3PO4
This means that the parent ion was doubly charged which is why the h3PO4 has is mass halved (m/2Z)
How can you detect the exposure of the amino acids to the solvent
If they do hydrogen deuterium exchange when in the deuterium solvent
Which HD exchange of the proton in amino acids cant and can be measured and why
The h on any carbon in the peptide is too slow to measure so can’t
H on the r groups too fast so can’t
But the amide H (N-H) is the correct speed so can measure
How is the analysis of D2O exhange done
D2O in solution coats the protein surface
Have to stop the reaction by dropping the temp
Put it in an acidic solution with high proton concentration
Digest the protein with pepsin (works at low pH)
How do you analyze the D2O exchange in a mass spec after doing the process
If no exchange, m/z is lower than if there is exchange
This is because d2O is heavier than the hydrogen
How can you check if a protein is bound to another protein using H/D exchange
For example there is a protein peruloside A
It binds and covers sequences of alpha and beta tubulin.
When the peruloside A is bound, the D2O exchange is lower since it’s protecting that area
How did they show on a ribbon diagram where the peruloside a binds
In the ribbon diagram, the red area show reducing in D2O labeling
And blue is increase in Labeling
So wherever less labelling is where the protein binds and protects those residues from exchange
