5.1 Peptide mass fingerprinting and de novo sequencing

Peptide mass fingerprinting and de novo sequencing are powerful tools for protein identification. PMF uses peptide masses to match proteins in databases, while de novo sequencing determines peptide sequences from scratch using MS/MS data.

Both techniques have unique strengths and limitations. PMF excels in rapid protein identification for well-characterized organisms, while de novo sequencing shines in discovering novel proteins and analyzing unsequenced organisms.

Peptide Mass Fingerprinting

Principles of peptide mass fingerprinting

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• Peptide mass fingerprinting (PMF) identifies proteins by analyzing peptide fragment masses using mass spectrometry
• Process involves digesting protein samples with specific enzymes (trypsin) to generate peptides for mass spectrometry analysis
• Experimental peptide mass spectrum compared to theoretical spectra from protein databases for identification
• PMF effectively identifies proteins in complex mixtures, validates recombinant protein expression, and characterizes post-translational modifications (phosphorylation, glycosylation)

Process of de novo sequencing

• De novo sequencing determines peptide sequences without prior knowledge using tandem mass spectrometry (MS/MS) data
• Steps include:
  1. Peptide fragmentation in mass spectrometer
  2. Analysis of fragment ion spectra
  3. Interpretation of mass differences between fragment ions
  4. Identification of amino acid residues from mass differences
  5. Assembly of complete peptide sequence
• Technique analyzes proteins absent from databases, discovers new protein variants (splice variants), and characterizes proteins from unsequenced organisms (extremophiles)

Peptide mass fingerprinting vs de novo sequencing

• Data input: PMF uses peptide masses from MS spectra, while de novo utilizes fragment ion spectra from MS/MS
• Database dependency: PMF relies heavily on protein sequence databases, de novo works independently
• Sequence information: PMF provides protein identification without direct sequence, de novo generates actual peptide sequences
• Analysis complexity: PMF generally simpler and faster, de novo more complex and computationally intensive

Advantages and limitations in research

• PMF advantages: Rapidly identifies proteins in well-characterized organisms (human, mouse), suitable for high-throughput analysis, uses less expensive and simpler instrumentation
• PMF limitations: Less effective for complex protein mixtures, struggles with unsequenced organisms, limited ability to detect post-translational modifications
• De novo advantages: Identifies novel proteins and peptides, effective for unsequenced organisms (newly discovered species), detects and localizes post-translational modifications
• De novo limitations: Time-consuming and computationally intensive, requires high-quality MS/MS spectra, may struggle with longer peptides (>20 amino acids) or incomplete fragmentation