Mass Spectrometry for Proteomics

Summary

• Mass spectrometry (MS) is critical in proteomics for analyzing biological samples.
• It has advanced to address complex biological questions.
• New technologies like Orbitrap and electron transfer dissociation (ETD) enable novel proteomic applications.
• Bottom-up proteomics predominates, but middle- and top-down strategies provide better characterization of protein isoforms and PTMs.
• Stable isotope labeling has advanced MS from descriptive to dynamic measurement of protein changes.

Introduction

• Proteomics aims to characterize all proteins, including expression, localization, and PTMs.
• Proteome varies across cells unlike the genome.
• Mass spectrometry is preferred over other techniques (e.g., 2DE, two-hybrid analysis) for its depth in proteome analysis.
• MS applications in proteomics:
  • Cataloging protein expression
  • Defining protein interactions
  • Identifying protein modification sites

Mass Spectrometric Instrumentation

• MS measures mass-to-charge ratio (m/z) of ions.
• Components: Ion source, Mass analyzer, Detector.
• Electrospray ionization (ESI) and MALDI revolutionized protein analysis.
• Mass analyzers used in proteomics:
  • Quadrupole (Q)
  • Ion Trap (QIT, LTQ)
  • Time-of-Flight (TOF)
  • Fourier-transform ion cyclotron resonance (FTICR)
• Hybrid instruments combine capabilities (e.g., Q-Q-Q, Q-TOF).
• Recent advances: Orbitrap for high resolution and accuracy.

Table 1: Performance of Mass Spectrometers

• Highlights mass resolution, accuracy, sensitivity, application.

LTQ-Orbitrap

• Combines LTQ's robustness with Orbitrap's accuracy.
• Capable of very high mass resolution and accuracy.

Fragmentation Methods

• Tandem MS (MS/MS) crucial for sequencing and PTM analysis.
• Collision-induced dissociation (CID) is traditional but has limitations for large peptides and intact proteins.
• Electron-capture dissociation (ECD) and ETD provide better fragmentation coverage.
  • ECD is nonergodic, preserving labile PTMs.
  • ETD extends ECD-like fragmentation to benchtop mass spectrometers.

Proteomic Strategies

• Bottom-up Proteomics: Standard for complex sample analysis.
  • Digestion followed by peptide analysis.
  • Commonly used in shotgun proteomics.
• Top-down Proteomics: Analyzes intact proteins for complete characterization.
  • More effective for analyzing protein isoforms and PTMs.
  • Challenges include analytical throughput and complexity.

Quantitative Proteomics

• Uses stable isotopes for measuring dynamic changes in protein/PTM abundances.
• Metabolic Labeling (e.g., SILAC) and Post-biosynthetic Labeling.
• Stable isotope-labeled synthetic peptides allow absolute quantification.

Conclusions and Outlook

• MS has become indispensable in understanding protein expression, interaction, and modification.
• Despite advances, challenges remain in sensitivity, dynamic range, and PTM complexity.
• Future efforts will focus on improving technology and data utilization.

Acknowledgements

• Funded by National Institutes of Health and American Cancer Society.

References

• Cites significant literature on MS advancements and applications.