Proteomics is the large-scale study of proteins, their structures, functions, interactions, and modifications. Unlike the genome, which is relatively static, the proteome is dynamic and reflects the cell’s state, with protein expression, localization, modification, and turnover all subject to regulation.
Mass Spectrometry Principles
Mass spectrometry measures the mass-to-charge ratio of ionized molecules. A mass spectrometer has three essential components. The ion source converts molecules into gas-phase ions. The mass analyzer separates ions by their mass-to-charge ratio. The detector measures the abundance of each ion. Proteins and peptides are typically analyzed using electrospray ionization or matrix-assisted laser desorption ionization.
Electrospray ionization produces multiply charged ions from peptides in solution, generating a series of peaks from which the molecular mass can be calculated. MALDI typically produces singly charged ions from peptides incorporated into a crystalline matrix and desorbed by a laser pulse. Both methods are soft ionization techniques that preserve the integrity of the analyte.
Mass Analyzers
Several mass analyzer types are used in proteomics. Quadrupole mass filters transmit ions of a selected mass-to-charge ratio through four parallel rods with oscillating electric fields. Time-of-flight analyzers measure the time ions take to travel a fixed distance, with lighter ions arriving faster. Orbitrap analyzers trap ions in an electrostatic field and measure their oscillation frequencies, providing high resolution and mass accuracy. Ion trap analyzers confine ions in a three-dimensional electric field and sequentially eject them. Hybrid instruments combining multiple analyzer types, such as quadrupole-Orbitrap or triple quadrupole instruments, provide flexibility for different experimental strategies.
Bottom-Up Proteomics
Bottom-up proteomics analyzes proteins (often separated by SDS-PAGE) after digestion into peptides, typically using trypsin, which cleaves after arginine and lysine residues. The resulting peptide mixture is separated by liquid chromatography and analyzed by tandem mass spectrometry. In data-dependent acquisition, the mass spectrometer selects the most abundant peptide ions for fragmentation by collision-induced dissociation. The resulting fragment ion spectra are searched against protein sequence databases to identify the peptides and their parent proteins.
Tandem Mass Spectrometry
In tandem mass spectrometry, a precursor ion of a specific mass-to-charge ratio is selected and fragmented, and the fragment ion masses are measured. Collision-induced dissociation fragments peptides primarily at amide bonds, producing b and y ions. The mass difference between adjacent y ions or b ions reveals the amino acid sequence. High-energy fragmentation methods such as electron-transfer dissociation provide complementary information, preserving labile post-translational modifications.
Protein Identification
Protein identification from tandem mass spectra uses database search engines such as Mascot, Sequest, or MaxQuant. The observed fragment ion masses are compared with theoretical spectra generated from in silico digestion of protein sequences in a database. Statistical analysis determines the confidence of each identification. De novo sequencing, which derives the peptide sequence directly from the mass spectrum without a database, is used for organisms with unsequenced genomes or for identifying novel peptides.
Quantitative Proteomics
Quantitative proteomics measures changes in protein abundance between conditions. Label-based methods introduce stable isotope tags that are distinguished by mass shifts. SILAC incorporates isotopically labeled amino acids through metabolic labeling in cell culture. TMT and iTRAQ use chemical tags that release reporter ions during fragmentation, allowing multiplexed quantification of up to 16 samples. Label-free quantification compares peptide ion intensities or spectral counts across runs, requiring careful normalization.
Post-Translational Modification Analysis
Mass spectrometry is uniquely suited for identifying and localizing post-translational modifications. Phosphorylation is detected by a characteristic mass increase of 80 Da and a neutral loss of phosphoric acid during fragmentation. Enrichment methods such as immobilized metal affinity chromatography or titanium dioxide chromatography are used to purify phosphopeptides before analysis. Glycosylation is more challenging because glycopeptides fragment poorly and glycan structures are heterogeneous. Specialized fragmentation methods such as electron-transfer dissociation preserve glycan-peptide linkages, enabling site-specific analysis.
Clinical Proteomics
Clinical proteomics applies proteomic technologies to medical problems. Biomarker discovery identifies proteins whose abundance changes in disease, potentially enabling early diagnosis or treatment monitoring using techniques such as ELISA. Tissue proteomics analyzes formalin-fixed, paraffin-embedded clinical specimens. Plasma proteomics faces challenges due to the enormous dynamic range of protein concentrations, with albumin alone constituting more than half of total protein. Affinity depletion of abundant proteins and extensive fractionation are typically required for deep plasma proteome analysis.
