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Biochemistry > Mass Spectrometry > Flashcards

Mass Spectrometry Flashcards

1
Q

What Information does Mass Spectrometry Provide?

A

Measuring the molecular weight (MW) of a sample.

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2
Q

What does a mass spectrometer do?

A
  • produces charged particles from chemical substances
  • uses electric and magnetic field to measure the mass “weight”
    of the charged particles
    can also give structural information.
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3
Q

How does a mass spectrometer work?

A

Source: - ions are produced - ions are easier to manipulate than neutral molecules.

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4
Q

What does the analyser do in a mass spectrometer?

A

separates ions according to mass (m) to charge (z) ratio (m/z). Most can be as accurate as 0.01% of m/z. Monitors under high vacuum. (<10-5 mBar)

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5
Q

What does a detector do in a mass spectrometer?

A

produces a signal for the separated ions (m/z spectrum) monitors the ion current e.g. photomultiplier. Monitors under high vacuum. (<10-5 mBar)

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6
Q

Name 3 different sources

A

Electrospray ionisation, Matrix Assisted Laser Desorption Ionisation (MALDI), Electron Impact

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7
Q

How are samples converted into gas phase molecules?

A

Under high vacuum (<10-5 mbar). Can generate either positively or negatively charged ions as they easier to detect than neutral molecules.

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8
Q

Explain electrospray ionisation

A

Contains 50uM capillary. Releases droplet containing ions. As droplet evaporates the electric field increases and ions move towards the surface, ions evaporate from the surface

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9
Q

What are the conditions (atmosphere, voltage, diameter) of electrospray ionisation when the sample is in solution?

A

1atm. High voltage applied to metal sheath: 4kV. Inner tube diameter: 100um. N2 gas.

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10
Q

What is the voltage for MALDI? What type of light goes in? What is the sample like?

A

30,000V. Pulsed laser light UV or IR. Sample is co-crystallised with the matrix on a target plate.

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11
Q

Why does the mass analyser operate under high vacuum?

A

Keeps ions bumping into gas molecules.

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12
Q

Which are the most common mass analysers for bioanalysis?

A

Quadrupole, time-of-flight (TOF) and ion traps.

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13
Q

What happens after ions are repelled out of the ion source?

A

Accelerated towards the analyser, analyser uses electric field to cause deflecting force on the molecule.

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14
Q

What does deflection in the mass spec analyser depend on?

A

The strength of field and mass to charge of the ion.

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15
Q

What can cause field and focusing on the detector results in a mass spec?

A

Changing strength of field and focussing.

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16
Q

How does quadrupole work?

A

4 parallel metal rods. Applied voltages affect the trajectory of ions travelling between the 4 rods. Only ions of a certain m/z ratio pass through the filter. All other ions are thrown out of their original path. Mass spec obtained by monitoring ions passing through the quadrupole filter as the voltages on the rods are varied. Quadrupoles have variable ion transmission modes.

17
Q

How is structural information derived? What procedure is this for?

A

Interpretation of MS/MS, by fragmenting the sample and analysing the products generated. The detection of specific drug metabolites in biological matrices.

18
Q

What is MS/MS?

A

Using 2 mass analysers (combined in 1 instrument) to select an analyte (ion) from a mixture then generate fragments from it to give structure info.

19
Q

What information can a parent ion provide when fragmented?

A

Structural information.

20
Q

How is the sample affected in LC/MS/MS?

A

Can introduce the sample directly or use a chromatographic technique.

21
Q

Why are compounds in liquid phase eluted from the HPLC at atmospheric pressure?

A

As mass spec accepts ‘only’ gas phase ions at high vacuum.

22
Q

What 2 things does the ion have to be in LC/MS/MS?

A

liquid —> gas

neutral —> ion

23
Q

How does the solvent delivery system work in HPLC?

A

Pushes the solvent stream through the instrument at constant flow rate.

24
Q

How does the autosampler work in HPLC?

A

Introduce the sample into the liquid stream of the instrument.

25
Q

Describe the ‘column’ in HPLC.

A

A stainless steel tube packed with silicon beads that separates compounds.

26
Q

How does the detector in HPLC work?

A

An optical sensor that detects changes in the characteristics of the solvent stream.

27
Q

What is the data system in HPLC?

A

A means of controlling the system components and storing, processing and displaying data.

28
Q

What does SACDD stand for and what does it describe?

A

Solvent delivery system. Autosampler. Column. Detector. Data system. Describes HPLC.

29
Q

What must the solvents be like in LC/MS and give 4 examples.

A

Volatile. Water, acetonitrile, methanol, acetate.

30
Q

What are 3 examples of non-volatile solvents?

A

Phosphates, borate, citrate.

31
Q

What will be the X, Y and Z axis on a 3D map for HPLC analysis?

A

X = Time Y = Intensity Z = Wavelength

32
Q

What are mass measurements good at identifying, verifying and quantitating?

A

Pharmaceutical, environmental, geological, biotechnology.

33
Q

Why is mass measurement good for pharmaceuticals?

A

Drug discovery, combinational chemistry drug metabolism metabolites.

34
Q

Why is mass measurements good environmentally and geologically?

A

PAHs, water quality, food contamination. Geo = oil composition.

35
Q

Why is mass measurement good for biotechnology?

A

Recombinant proteins, proteins isolated from natural sources, peptides, oligonucleotides, drug candidates, synthetic organic chemicals, polymers.

36
Q

What can be used to study proteomics?

A

2D gel electrophoresis.

37
Q

How accurate is the molecular weight of large biomolecules measured to within and what is this sufficient for?

A

0.01% of the total molecular weight of the sample. This is sufficient to allow minor mass changes to be detected e.g. the substitution of 1 amino acid for another.

38
Q

What is an advantage and disadvantage of MALDI-TOF?

A

A = many proteins at once. D = difficult to quantitate.

39
Q

Name 4 techniques interfaced with mass spectrometry. Name 2 more that involve direct injection.

A

Gas, Liquid and Capillary Liquid chromatography. Capillary Electrophoresis. Direct injection: Thin layer chromatography. Proteomics (2D gels).

Biochemistry (7 decks)

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