1. Sample Introduction to MS Systems:

Transcription

1 MS Overview: Sample Introduction to MS Systems: Chromatography Interfaces: Electron impact: Used mainly in Protein MS hard ionization source Electrospray Ioniztion: Used extensively in protein MS ESI Continued: Example of an ESI-MS of Cytochrome C Matrix Assisted Laser Disorption-Ionization (MALDI) Matrix Assisted Laser Disorption-Ionization (MALDI) Graphic An example of MALDI-TOF analysis of a 2D-PAGE separation Mas Analyzers Quadrupole Analysers: Function of DCA and AC bias An example of a quadrupole Mass Spectrum of a Protein Digest Magnetic and electric sector analyzers: Electric plus magnetic sectors for high resolution: Time of Flight Analyzers Ion Trap Mass Analyzers Ion Trap Mass Analyzers Ion Cyclotron Resonance Mass Analyzers MS-MS with the triple quadrupole Modes of MSMS using TRIPLE-QUAD Explanation of MSMS modes Hard vs. soft ionization Resolution defined The need for high resolution....26

2 1. Sample Introduction to MS Systems:

3 1.1. Chromatography Interfaces:

4 1.2. Electron impact: Used mainly in Protein MS hard ionization source.

5 1.3. Electrospray Ioniztion: Used extensively in protein MS

6 ESI Continued:

7 Example of an ESI-MS of Cytochrome C

8 1.4. Matrix Assisted Laser Disorption-Ionization (MALDI) Gives predominance of M + sample in vacuum chamber proteins in matrix matrix ionizes supersonic expansion

9 Matrix Assisted Laser Disorption-Ionization (MALDI) Graphic 2.

10 An example of MALDI-TOF analysis of a 2D-PAGE separation.

11 2. Mas Analyzers

12 2.1. Quadrupole Analysers:

13 Function of DCA and AC bias

14 An example of a quadrupole Mass Spectrum of a Protein Digest.

15 2.2. Magnetic and electric sector analyzers:

16 2.3. Electric plus magnetic sectors for high resolution: Electric Sector Focuses: Speed distribution Magnetic Sector Re-focuses: Directional distribution At double-focus point, all ions have narrow speed and direction distributions, so only ions of a given M/z exit at that point. What determines the M/z values that exit? E-field on electric sector B-field on magnetic sector R attainable: 100,000

17 2.4. Time of Flight Analyzers

18 2.5. Ion Trap Mass Analyzers

19 Ion Trap Mass Analyzers

20 2.6. Ion Cyclotron Resonance Mass Analyzers

21 3. MS-MS with the triple quadrupole. Product ion scanning produces a fingerprint for a given peptide.

22 3.1. Modes of MSMS using TRIPLE-QUAD

23 3.2. Explanation of MSMS modes. Mode Characteristic m/z scans vs time Product Ion Scanning Precursor Ion Scanning Neutral Loss Scanning Multiple Ion Monitor In this mode one filters the sample and scan reaction products. This will produce a fingerprint for all eluent that generate the Q1 mass. One can fish for a given target or targets and see the fingerprints for all ions of a given mass. May generate multiple ID s potentially generating an ID Gives you the MASS spectrum of an ion that generates a given single mass product. Used, for example, if you want to inventory the proteints that are phosphorylated or glycosylated protein. The m/z axis is for Q1, but the signal is for the Q3 mass only. Therefore, what you learn are the m/z values that yield a product with a given m/z. The fixed difference between the analyzers dramatically reduces the complexity of the output. The signal is reduced to exclusively detect those ions that lose a certain mass. You won t know the parent mass, only the time that it shows up. So, this must also rely on knowledge of the chromatographic retention time for that ion. Similar to neutral loss in the sense that it is highly selective. Note also that this is essentially a highly selective chromatographic detector! Both the initial mass and the mass loss are scanned. You may realistically detect only a single protein in a very complex mixture this way. I vs time Information Scans for fragment fingerprint of peptides of a given mass. Tells you the Q1 m/z of all peptides that yield a given fragment. Tells you the retention time at which something of unknown m/z elutes that loses a fixed mass. Tells you the retention time at which a very specific entity elutes that yields a fixed Q1 -collision - Q3 product.

24 4. Hard vs. soft ionization.

25 5. Resolution defined. Unfortunately, the word resolution is commonly and loosely used to express both the quantities Δm and m/δm. High resolution refers to a large m/δm value for a given m value. A purist would call this high resolving power. I m too old to change though, and I will still refer to instruments as high resolution when they are really high resolving power.

26 5.1. The need for high resolution.