De Novo Protein Sequencing
Principle of De Novo Protein Sequencing
De novo protein sequencing determines the amino acid sequence of proteins without relying on existing DNA or protein databases. This technique is based on the predictable fragmentation patterns of peptides during mass spectrometry. Specific cleavage patterns are identified, and the mass differences between peaks in the spectrum are used to deduce amino acid sequences and identify post-translational modifications. During fragmentation, peptides produce various ion types. Cleavage near the N-terminal generates a, b, and c ions, while cleavage near the C-terminal produces x, y, and z ions. Among these, b ions are particularly significant as they result from bond cleavage between two amino acid residues. The mass difference between adjacent b ions corresponds to the mass of an amino acid residue. By analyzing these mass differences, the R group mass and corresponding amino acid identity can be determined.
Methods for De Novo Protein Sequencing
1. Sample Preparation
(1) Protein Extraction and Purification: Proteins are extracted from biological samples (e.g., cells, tissues, or body fluids) and purified using methods such as centrifugation, chromatography, or electrophoresis. This step enhances protein purity (typically ≥80%-90%) and minimizes interference during analysis.
(2) Protease Digestion: Proteins are enzymatically cleaved into smaller peptide fragments using proteases such as trypsin or chymotrypsin. Employing multiple proteases ensures comprehensive sequence coverage.
2. Mass Spectrometry Analysis
(1) Ionization: Peptide fragments are ionized using techniques like electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI). Ionization imparts a charge to peptides, enabling analysis in the mass spectrometer.
(2) MS1 Analysis: The ionized peptides are first subjected to MS1 analysis to determine the mass-to-charge ratio (m/z) of precursor ions.
(3) Collision-Induced Dissociation (CID): Precursor ions are fragmented through collisions with inert gases (e.g., helium, argon), generating fragment ions.
(4) MS2 Analysis: Fragment ions are further analyzed in MS2 to provide detailed peptide fragmentation data.
3. Data Analysis
(1) Data Processing: Software tools like PEAKS or Mascot are used to filter noise, classify mass-to-charge ratios, and analyze MS2 data.
(2) Sequence Deduction: Peptide sequences are deduced by analyzing mass differences between fragment ions and considering potential post-translational modifications.
(3) Sequence Validation: To ensure accuracy, deduced sequences are validated using complementary methods like Edman degradation or synthetic peptide comparisons.
Applications and Limitations
1. Applications
De novo sequencing is vital for identifying novel proteins in fields like microbiology, botany, and biopharmaceuticals. For example, novel enzymes discovered in microbial strains can be sequenced to facilitate functional research and development.
2. Limitations
The process requires high-quality mass spectrometry data. Low resolution or excessive interference can compromise accuracy. Long peptides (>20-30 amino acids) are particularly challenging due to increased complexity in fragmentation patterns.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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