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Introduction
Mass spectrometry (MS) is indispensable for peptide characterization. This powerful technique provides molecular weight confirmation, sequence information, and structural details that complement chromatographic purity data.
Fundamentals of Mass Spectrometry
How Mass Spectrometry Works
Mass spectrometry measures the mass-to-charge ratio (m/z) of ions:
- Ionization: Sample molecules are converted to gas-phase ions
- Mass Analysis: Ions are separated by m/z ratio
- Detection: Ion abundance is measured
- Data Processing: Spectra are interpreted to identify compounds
Key Terms
- Molecular ion: Ion corresponding to intact molecule
- m/z: Mass-to-charge ratio
- Resolution: Ability to distinguish close masses
- Mass accuracy: How close measured mass is to true mass
Ionization Techniques
Electrospray Ionization (ESI)
ESI is the most common ionization method for peptides.
Process:
- Solution sprayed through charged needle
- Droplets evaporate, leaving charged molecules
- Multiple charge states observed for larger peptides
Advantages:
- Gentle ionization preserves structure
- Compatible with LC coupling
- Works well for polar peptides
Considerations:
- Multiple charge states can complicate spectra
- Sensitive to buffer composition
- Ion suppression possible with mixtures
Matrix-Assisted Laser Desorption/Ionization (MALDI)
MALDI uses a laser to ionize samples mixed with matrix.
Process:
- Sample co-crystallized with UV-absorbing matrix
- Laser pulse desorbs and ionizes sample
- Primarily singly-charged ions observed
Advantages:
- Simple spectra (usually single charge state)
- Tolerates salts and buffers
- Good for higher molecular weight peptides
Considerations:
- Matrix interference at low mass
- Less quantitative than ESI
- Typically offline analysis
Mass Analyzers
Quadrupole
- Workhorse of routine analysis
- Good for targeted quantitation
- Unit mass resolution
Time-of-Flight (TOF)
- High mass range
- Fast analysis
- Good mass accuracy and resolution
Orbitrap
- Very high resolution
- Excellent mass accuracy
- Ideal for complex mixture analysis
Ion Trap
- MS/MS capability
- Good sensitivity
- Moderate resolution
LC-MS for Peptide Analysis
Why Couple LC with MS?
Combining liquid chromatography with mass spectrometry provides:
- Separation of complex mixtures
- Real-time mass confirmation
- Quantitation capabilities
- Impurity identification
LC-MS Configurations
Single Quadrupole
- Basic mass confirmation
- Selected ion monitoring (SIM)
- Cost-effective option
Triple Quadrupole (QqQ)
- Selected reaction monitoring (SRM)
- Superior quantitation
- High selectivity
Q-TOF
- High resolution MS and MS/MS
- Accurate mass measurement
- Ideal for characterization
Orbitrap Systems
- Highest resolution available
- Excellent mass accuracy
- Multi-attribute methods
