Mass Spec 2

Question 1

What are the two ways to identify proteins

Which is better

Answer 1

Peptide mass fingerprinting

Shotgun sequencing (better)

Question 2

What is the mass redundancy problem

Answer 2

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

Question 3

What is tandem mass spectrometry (MS/MS)

Answer 3

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

Question 4

In tandem mass spec what is the shotgun

Answer 4

Shotgun is the thing that breaks the sample into fragments

So the shotgun is the inert gas he or ar

Question 5

How do you analyze the fragmentation of the peptide in MS/MS

Answer 5

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)

Question 6

How do you get AX

BY

CZ

From the MS/MS fragment

Answer 6

If fragmented in the c aplha and the c=o

In the c=o and N-H

In the N-H and C alpha

Question 7

Is MS/MS what fragments do we see

Answer 7

The one that keeps the positive charge that we selected it to have

Question 8

Even though many fragments are made in MSMS which do we focus on

Answer 8

The B and Y ions (fragmented between the c=o and N-H

Question 9

How do you find the mass of the B type ion and the y type

Answer 9

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

Question 10

How can you check for error when finding the mass of the y and B type ion

Answer 10

The sums of their masses should equal the total mass of the peptide + 2 (because 2 protons)

Question 11

What residues are hard to distinguish if we are checking their m/z ratio

Answer 11

Leucine and isoleucine because isomers

Question 12

How do you analyze a mama spectrum

Answer 12

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

Question 13

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

Answer 13

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)

Question 14

What is the sequence tag approach of MSMS

Answer 14

You have the entire mass of the peptide and I little bit of sequence info

You don’t have the full sequence

Question 15

What is the key thing that allows MSMS to work

Answer 15

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

Question 16

What are the applications of mass spec

Answer 16

Postranslational modification

Deconvoluted soectra

Structural biology

Question 17

What is a deconvoluted spectrum

Answer 17

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

Question 18

What is RNAse B

Answer 18

The glycosylated form of RNase A

Question 19

How can a mass spec show us the glycosylation of a protein

What is special about it

Answer 19

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

Question 20

How can you tell if something was shotgun sequenced (MSMS)

Answer 20

There are B and Y type ions in the spectrum

Question 21

How can shotgun sequencing show is phosphorylation of a protien

Answer 21

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)

Question 22

How can you detect the exposure of the amino acids to the solvent

Answer 22

If they do hydrogen deuterium exchange when in the deuterium solvent

Question 23

Which HD exchange of the proton in amino acids cant and can be measured and why

Answer 23

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

Question 24

How is the analysis of D2O exhange done

Answer 24

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)

Question 25

How do you analyze the D2O exchange in a mass spec after doing the process

Answer 25

If no exchange, m/z is lower than if there is exchange

This is because d2O is heavier than the hydrogen

Question 26

How can you check if a protein is bound to another protein using H/D exchange

Answer 26

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

Question 27

How did they show on a ribbon diagram where the peruloside a binds

Answer 27

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