Mass Spec 1

Question 1

What is mass spectrometry

Answer 1

It’s used to determine the molecular weight of molecules using the mass to charge ratio (m/z)

Question 2

How are the ions from mass spec formed

Answer 2

From the gas phase of the molecule

When a proton is added you get a positive ion

When proton is removed you get a negative ion

Question 3

How can the charged ion from mass spec be analyzed

Answer 3

By electric and/or magnetic fields

(Ex gel electrophoresis)

Question 4

What are the components of a mass spec

Answer 4

The ion source: makes ion in the gas phase

The mass analyzer: separate the ion according to their m/z

The detector: collect the ion and amplifies their signals

The data system: controls the mass spec and stores/analyzes the data

Question 5

What are the three characteritics of mass spec

Answer 5

Destructive technique: the sample isn’t recovered

Gas phase: the ionized sample flies

High resolution technique: small differences in mass can be measured

Question 6

If you have a formula ex. C3H7NO2 how do you find the nominal, monoisotopic , and averaged mass

Answer 6

Nominal: 3x12 + 7x1 + ….

Monoisotopic: same as nominal but use the exact mass instead

Average: same as nominal but use the average mass instead

Question 7

How do you find the average mass of an atom

Answer 7

Multiply exact mass of each isotope by the decimal percentage of that isotope

Then add the two values

DOUBLE CHRCK

Question 8

What is a mass spectrum a plot of

Answer 8

Signal intensity vs m/z

Bigger peak means more abundant (so signal intensity is proportional to abundance)

Question 9

Generally what is the exact mass of the most abundant isotope in comparison to the average mass

Answer 9

The most abundant has its mass low than the average mass

Ex. C13 is 12 but average is 12.0015

Question 10

What is monoisotopic mass

Answer 10

Measured using the exact mass of the most abundant isotope

Question 11

What is average mass

Answer 11

It’s the mass weighted with the abundance

Question 12

What is the equation for mass accuracy and what is it expressed as

Answer 12

(True mass - obs / true ) x 10^6

Question 13

In general what is a mass spectrum plot axis

What does it look like

Answer 13

Signal intensity vs m/z

In contrast to a chromatogram, It has sharp peaks

Question 14

What does all organic matter have a distribution of

Answer 14

Isotopes

Question 15

What is the effect of natural abundance

Answer 15

Different isotopes become relevant in a mass spectrum with increasing abundance of that element

Question 16

How can you find the average mass of a compound through the mass spectrum

Answer 16

You have many peaks at different masses

If you get a low resolution curve of these peaks, you can get the average mass from the centre of it

If it’s a low resolution curve the average mass you be closer to the monoisotopic mass

Question 17

What is accuracy expressed as

Which is best for mass spec

What do you usually get from the equipment for proteomics

What does this tell us

Answer 17

ppm

0.1ppm

10-50ppm

To identify proteins we need tools like MS so get high accuracy

Question 18

If you have 20% accuracy how many ppm is this

Answer 18

200,000ppm

Question 19

What gives a better mass spectrum, a protien mixture or peptides

Answer 19

Peptides

They give different resolutions

Peptides give the average mass not the monoisotopic mass

They are less resolved

Question 20

In getting the ion source for mass spec what type of ionization is it and what are they

Answer 20

There is soft ionization

MALDI

ESI

Question 21

What mass analyzers do we use in mass spec

Answer 21

time of flight (TOF)

Quadrupole

Ion trap

FT ICR

Question 22

What is MALDI

Answer 22

You have your analyte (protein or peptides) on a MALDI plate in a sample matrix

The matrix absorbs UV light from the laser source and transfers a proton to the analyte in the matrix

When the analyte gets a positive charge, it repels and desorbs off from the matrix

Then the postive particles are now ionized

Question 23

In doing a MALDI tof experiment what do you see in the spectra

What do you make sure

Answer 23

You see the peptide ion (M+H)+

But you also see ions that flew to the detector from the matrix

Make sure that the matrix ion are separate from the peptide ion

Question 24

What is ESI

Answer 24

The liquid sample is in a needle

The liquid is either basic or acidic (negative or postive charged)

The needle gets a voltage applied to it (if sample positive charge apply positive to negative voltage)

that makes the charged molecules in the liquid leave the needle in droplets

To get the sample as a gas we apply a nebulizer (nitrogen gas)

Then the concentrated charged dry ions reach the mass spec

Question 25

What does the electrospray mass spectrum give us

Answer 25

Multiple peaks with different number charges Every peak shown is smaller than if it was one peak This increases the accuracy but decreases the signal intensity

Question 26

What is the equation to find the mass of a peak in ESI (Mr)

Answer 26

mi = Mr + i / i And mi +1 = Mr + i + 1 / i + 1 Where i is the charge

Question 27

What are the experimental difficulties in getting the mass of a protein

Answer 27

The protein have to be clean so No salt (won’t fly well in the mass spec) No Detergent (SDS) No buffer (have to use a volatile buffer that won’t stay like ammonium carbonate and ammonium sulfate) No polyethylene glycol (normally used to concentrate the protein)

Question 28

What is mass resolution in mass spec

Answer 28

ratio of the mass of the peak / its width at half its max intensity R= m / delta m

Question 29

What is the process of identifying proteins using mass spectrometry

Answer 29

Separate the proteins using a 1D , 2D gel (separating by charge then size) or liquid chromatography Cleave the proteins with trypsin Then do one or two mass spec analysis: PMF Tandem mass spec

Question 30

What is the concept of PMF

Answer 30

You can use the predicted protein sequences from genome sequencing The proteins you measure don’t have to be previously sequenced, only the open reading frame of the gene needs to be known

Question 31

What is the process of PMF

Answer 31

purify the protien mixture to get single protein samples Cleave the protien into peptides using protease (chymptrypsin or trypsin) Use mass spec to get the mass of every peptide Then compare it to published data and identify the protein that has these peptides masses

Question 32

What does chymotrypsin cleave What about trypsin

Answer 32

Chemotrypsin cleave after hydrophobic residues Trypsin cleaves after argenine or lysine

Question 33

If the trypsin did a missed cleavage and did not cleave after R or K what happens

Answer 33

Since it would have two peptides instead of the one we expect, you get a bigger mass than expected

Question 34

What is trypsin auto proteolysis and what should we see

Answer 34

The trypsin cleaves itself and gives its own peaks in the PMF spectrum These peaks should always be in the same place

Question 35

Once you get the peptides at different masses in PMF what can you do

Answer 35

You can compare these masses to those in the protein data bank and it can predict the protien that you have based on the matches of MW

Question 36

What are uniques peptides and how can they help us in analysis of protiens What is coverage percent and how does it help in analysis of protiens

Answer 36

Coverage percent is the amount of the protien that has been matched using the peptides , this helps in confidence in our analysis and identification of the protien since more of the protien was covered Number of Unique peptides are peptides we found during mass spec that match only to a specific protien . Having more of these lets us make sure our protien we’ve identified our protien , lead to less false postive for identification