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Bcem 431 > Mass Spec 1 > Flashcards

Mass Spec 1 Flashcards

1
Q

What is mass spectrometry

A

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

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2
Q

How are the ions from mass spec formed

A

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

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3
Q

How can the charged ion from mass spec be analyzed

A

By electric and/or magnetic fields

(Ex gel electrophoresis)

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4
Q

What are the components of a mass spec

A

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

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5
Q

What are the three characteritics of mass spec

A

Destructive technique: the sample isn’t recovered

Gas phase: the ionized sample flies

High resolution technique: small differences in mass can be measured

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6
Q

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

A

Nominal: 3x12 + 7x1 + ….

Monoisotopic: same as nominal but use the exact mass instead

Average: same as nominal but use the average mass instead

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7
Q

How do you find the average mass of an atom

A

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

Then add the two values

DOUBLE CHRCK

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8
Q

What is a mass spectrum a plot of

A

Signal intensity vs m/z

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

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9
Q

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

A

The most abundant has its mass low than the average mass

Ex. C13 is 12 but average is 12.0015

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10
Q

What is monoisotopic mass

A

Measured using the exact mass of the most abundant isotope

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11
Q

What is average mass

A

It’s the mass weighted with the abundance

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12
Q

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

A

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

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13
Q

In general what is a mass spectrum plot axis

What does it look like

A

Signal intensity vs m/z

In contrast to a chromatogram, It has sharp peaks

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14
Q

What does all organic matter have a distribution of

A

Isotopes

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15
Q

What is the effect of natural abundance

A

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

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16
Q

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

A

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

17
Q

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

A

ppm

0.1ppm

10-50ppm

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

18
Q

If you have 20% accuracy how many ppm is this

A

200,000ppm

19
Q

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

A

Peptides

They give different resolutions

Peptides give the average mass not the monoisotopic mass

They are less resolved

20
Q

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

A

There is soft ionization

MALDI

ESI

21
Q

What mass analyzers do we use in mass spec

A

time of flight (TOF)

Quadrupole

Ion trap

FT ICR

22
Q

What is MALDI

A

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

23
Q

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

What do you make sure

A

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

24
Q

What is ESI

A

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

25
Q

What does the electrospray mass spectrum give us

A

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

26
Q

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

A

mi = Mr + i / i

And

mi +1 = Mr + i + 1 / i + 1

Where i is the charge

27
Q

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

A

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)

28
Q

What is mass resolution in mass spec

A

ratio of the mass of the peak / its width at half its max intensity

R= m / delta m

29
Q

What is the process of identifying proteins using mass spectrometry

A

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

30
Q

What is the concept of PMF

A

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

31
Q

What is the process of PMF

A

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

32
Q

What does chymotrypsin cleave

What about trypsin

A

Chemotrypsin cleave after hydrophobic residues

Trypsin cleaves after argenine or lysine

33
Q

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

A

You get a longer peptide chain than you should have

34
Q

What is trypsin auto proteolysis and what should we see

A

The trypsin cleaves itself and gives its own peaks in the PMF spectrum

These peaks should always be in the same place

35
Q

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

A

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

36
Q

Slides 30-34

A

Idk

Bcem 431 (26 decks)

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