4.4 Tandem mass spectrometry (MS/MS) for protein sequencing

Tandem mass spectrometry is a powerful tool for protein analysis. It breaks down complex molecules into smaller fragments, allowing scientists to identify and study proteins in detail. This technique is crucial for understanding protein structures and functions.

MS/MS helps researchers uncover the secrets of proteins by examining their building blocks. By fragmenting peptides and analyzing the resulting pieces, scientists can determine amino acid sequences and spot important modifications, shedding light on protein behavior in living systems.

Fundamentals of Tandem Mass Spectrometry

Tandem mass spectrometry for proteins

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• Tandem mass spectrometry (MS/MS) performs two-stage mass analysis for structural elucidation of molecules
• MS/MS identifies peptide sequences and determines post-translational modifications in protein sequencing
• Process involves ionizing peptides, selecting precursor ions, fragmenting selected ions, and analyzing resulting fragment ions
• Offers high sensitivity and specificity allows analysis of complex protein mixtures (blood serum, cell lysates)

Collision-induced dissociation principles

• Collision-induced dissociation (CID) fragments precursor ions through collisions with neutral gas molecules (helium, nitrogen, argon)
• CID accelerates precursor ions, transfers kinetic energy to internal energy upon collision, causing bond cleavage and ion fragmentation
• Generates b-ions (N-terminal fragments) and y-ions (C-terminal fragments)
• CID efficiency depends on collision energy, gas pressure, and ion charge state

Interpretation of MS/MS spectra

• MS/MS spectrum plots mass-to-charge ratio (m/z) on x-axis and ion intensity on y-axis
• Fragment ion series include a, b, c ions (N-terminal) and x, y, z ions (C-terminal)
• Peptide sequence determination involves identifying b and y ion series, calculating mass differences between adjacent peaks, and matching to amino acid residue masses
• De novo sequencing uses manual interpretation or automated algorithms for sequence assignment
• Database searching compares experimental spectra with theoretical spectra, scoring and ranking peptide matches

Peptide fragmentation in protein analysis

• Protein identification employs peptide mass fingerprinting, sequence tag searching, and protein inference from peptide identifications
• Characterizes post-translational modifications (PTMs) by identifying modification sites and quantifying modified peptides
• Structural analysis determines disulfide bonds and identifies protein-protein interaction sites
• Enables quantitative proteomics through relative quantification (label-free methods) and absolute quantification (isotope-labeled standards)
• Applications span biomarker discovery, drug development, and systems biology research