Protein & Peptide Analysis by Mass Spectrometry Flashcards
Why by Mass Spectrometry?
- Cheaper, quicker and easier than alternatives
- Answers in hours/days vs weeks/months
- Very sensitive so small amounts required
- Sample not required to be as pure/homogenous as Edman. Not quite as sensitive as Westerns but not reliant on a possibly non-existent antibody and does not give false positives like Westerns can
- Precise and accurate unlike eg SDS-PAGE
Drawbacks of Mass Spectrometry
- MS instrumentation is expensive as is maintenance
- Requires highly trained operators and analysts
- Highly dependent on databases
- Very sensitive; double-edged sword – Keratin contamination can be an issue
- Good sample preparation is paramount
What does MS consist of ?
Ion Source: Hard & soft ionisation
- Electron Impact (EI)
- Electrospray Ionisation (ESI)
Mass Analysing
Detector
m/z is measured (monoisotopic vs average)
Very qualitative. Can be quantitative but ionisation efficiency is
highly variable so controls must be in place
Types of MS
Important Mass Spectrometry Concepts
Tuning:
Many settings that can be altered that will change the way ions will move through the instrument
Mass Accuracy:
How close a measurement is to the actual. Calibration and lock masses
improve this. Important for limiting number of false positives
Resolution:
How well nearby masses are separated. Usually involves longer scanning
time. Important for seeing nearly isobaric ions
Dynamic Range:
Ability of instrument to see low level ions in the same scan as very abundant ions. Particularly Important in protein work where the stoichiometry of PTMs is often several orders of magnitude lower than the unmodified counterpart
Mass Error
x 1,000,000 = error in ppm
A high number (positive or negative) indicates a high deviation from the expected. High and low is relative
and needs be decided in the context of the instrument and how well calibrated it is
Resolution
High Performance Liquid Chromatography
