Mass Spec 1 Flashcards
What is mass spectrometry
It’s used to determine the molecular weight of molecules using the mass to charge ratio (m/z)
How are the ions from mass spec formed
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
How can the charged ion from mass spec be analyzed
By electric and/or magnetic fields
(Ex gel electrophoresis)
What are the components of a mass spec
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
What are the three characteritics of mass spec
Destructive technique: the sample isn’t recovered
Gas phase: the ionized sample flies
High resolution technique: small differences in mass can be measured
If you have a formula ex. C3H7NO2 how do you find the nominal, monoisotopic , and averaged mass
Nominal: 3x12 + 7x1 + ….
Monoisotopic: same as nominal but use the exact mass instead
Average: same as nominal but use the average mass instead
How do you find the average mass of an atom
Multiply exact mass of each isotope by the decimal percentage of that isotope
Then add the two values
DOUBLE CHRCK
What is a mass spectrum a plot of
Signal intensity vs m/z
Bigger peak means more abundant (so signal intensity is proportional to abundance)
Generally what is the exact mass of the most abundant isotope in comparison to the average mass
The most abundant has its mass low than the average mass
Ex. C13 is 12 but average is 12.0015
What is monoisotopic mass
Measured using the exact mass of the most abundant isotope
What is average mass
It’s the mass weighted with the abundance
What is the equation for mass accuracy and what is it expressed as
(True mass - obs / true ) x 10^6
In general what is a mass spectrum plot axis
What does it look like
Signal intensity vs m/z
In contrast to a chromatogram, It has sharp peaks
What does all organic matter have a distribution of
Isotopes
What is the effect of natural abundance
Different isotopes become relevant in a mass spectrum with increasing abundance of that element
How can you find the average mass of a compound through the mass spectrum
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
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
ppm
0.1ppm
10-50ppm
To identify proteins we need tools like MS so get high accuracy
If you have 20% accuracy how many ppm is this
200,000ppm
What gives a better mass spectrum, a protien mixture or peptides
Peptides
They give different resolutions
Peptides give the average mass not the monoisotopic mass
They are less resolved
In getting the ion source for mass spec what type of ionization is it and what are they
There is soft ionization
MALDI
ESI
What mass analyzers do we use in mass spec
time of flight (TOF)
Quadrupole
Ion trap
FT ICR
What is MALDI
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
In doing a MALDI tof experiment what do you see in the spectra
What do you make sure
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
What is ESI
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
What does the electrospray mass spectrum give us
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
What is the equation to find the mass of a peak in ESI (Mr)
mi = Mr + i / i
And
mi +1 = Mr + i + 1 / i + 1
Where i is the charge
What are the experimental difficulties in getting the mass of a protein
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)
What is mass resolution in mass spec
ratio of the mass of the peak / its width at half its max intensity
R= m / delta m
What is the process of identifying proteins using mass spectrometry
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
What is the concept of PMF
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
What is the process of PMF
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
What does chymotrypsin cleave
What about trypsin
Chemotrypsin cleave after hydrophobic residues
Trypsin cleaves after argenine or lysine
If the trypsin did a missed cleavage and did not cleave after R or K what happens
Since it would have two peptides instead of the one we expect, you get a bigger mass than expected
What is trypsin auto proteolysis and what should we see
The trypsin cleaves itself and gives its own peaks in the PMF spectrum
These peaks should always be in the same place
Once you get the peptides at different masses in PMF what can you do
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
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
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
