General Mass Spectrometry in the Cores

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General Mass Spectrometryin the Cores
David M. Smalley
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Mass Spectrometer System
Protein Sample
Sample prep
Mass Analyzer Separates ions
Data System
Detection Device
Bioworks Excalibur SEQUEST Mascot In-house
TOF Quadropole Ion Trap FT (ICR) MS
MALDI ESI
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4 Mass Spec Instruments

• Applied Biosystems Voyager-DE Pro
• Thermo Electron Corp. LTQ
• Thermo Electron Corp. LTQ-FT
• Applied Biosystems QStar Elite

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Voyager-DE Pro
MALDI-TOF
Ion Source - MALDI Matrix Assisted Laser
Desorption Ionization Mass Analyzer - TOF
Time of flight
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Ion Source MALDI(Matrix Assisted Laser
Desorption Ionization)
Sample
matrix material (benzoic acid derivative)
Laser



Ionized Peptides
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Voyager-DE Pro
Sample
Matrix (benzoic acid derivative)
Spot on a plate
Release of Ions (1 charge)
Let dry
Laser
Place into instrument
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Analyzer is a TOF or Time-of-Flight
Ion Detector
Flight Tube
Laser
V
Ion Source
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Peptide Mass Fingerprinting
MALDI-TOF
Cut out
Trypsin Digestion
Peptide Mixture
Albumin
585 amino acids
22 peptides (10 plus amino acids)
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(No Transcript)
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MALDI-TOFApplications

• Samples
• Peptides Provides MW (high resolution)
• Protein Provides a MW (low resolution)
• Applications
• Protein Mass Fingerprinting
• Quality control for synthetic peptides
• Others. (Recombinant protein, cleavage sites,
  kinetic assays, protein modifications, etc.)

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Advantages/Disadvantages

• Advantages
• Quick and easy
• Inexpensive
• High Throughput
• Disadvantages
• Limited Structural Information
• For Peptide Mass Fingerprinting
• Protein must be pure
• Better for yeast or E. coli
• Problem with contamination.

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4 Mass Spec Instruments
Mass Analyzer
Ion Source

• Voyager-DE MALDI single TOF
• LTQ ESI tandem
• LTQ-FT ESI tandem
• QStar Elite ESI tandem

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ESI Electrospray Ionization
to mass analyzer
Peptide solution



V
LC
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Single Stage MS
Scan Mode
Mixture of Peptides
Ion Source
MS1
Scanning
Ion Intensity
Mass/charge (m/z)
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Tandem MS
Peptides
Ion Source
MS1
1 peptide
Fixed (on one m/z)
Collision Cell
Fragments of peptide
MS2
Scanning
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ACDECAGH K ACDECAG
HK ACDECA
GHK ACDEC AGHK ACDE
CAGHK ACD ECAGHK AC
DECAGHK A CDECAGHK
ACDECAGHK
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Tandem MS
Peptides
Ion Source
MS1
1 peptide
Fixed (on one m/z)
Collision Cell
Fragments of peptide
MS2
Scanning
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Tandem MS (Can be tandem in time)
Ion Trap

• Selects 1 peptide
• Fragments that peptide
• Determines the m/z for the fragments

Peptides
Ion Source
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4 Mass Spec Instruments
Mass Analyzer
Ion Source

• Voyager-DE MALDI single TOF
• LTQ ESI tandem LIT
• LTQ-FT ESI tandem LIT-FT
• QStar Elite ESI tandem qTOF

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LTQ

• LC-ESI-ion Trap
• Ion Trap Tandem in time

Explain MSn
protein mixture
Trypsin Digestion



MS MS2 MSn
LC/ESI
peptide mixture
Ion Trap
Figure from Schwartz, Michael W. Senko and John
E. P. Syka, Journal of the American Society for
Mass Spectrometry, 13(6)659-669 (2002).
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MSn
Peptides
Ion Source
MS1
1 peptide
Fixed (on one m/z)
Collision Cell
Fragments of peptide
MS2
Scanning
Collision Cell
Ion Intensity
MS3
Fragments of fragments
200
100
150
50
m/z
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LTQ

• Strengths
• Excellent throughput
• Good Sensitivity
• MSn capabilities
• Robust
• Weaknesses
• Poor mass accuracy/ low resolving power
• Weak for quantification
• Poor for detection of modifications
• Not well suited for iTRAQ

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LTQ-FT

• LTQ with another ICR analyzer
• Allows for excellent mass accuracy and resolving
  power
• Better dynamic range than the LTQ
• Also, much more expensive

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QStar

• LC-ESI - Quadropole TOF (QqTOF)

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QStar

• Good mass accuracy and resolving power
• Good dynamic range
• Better for quantification
• iTRAQ and phosphorylation

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Summary

• Explained the major components of a mass
  spectrometry system
• Described the mass spectrometers in the MS core
  in sufficient detail for today's talks.