Question

The determination of the mass of a protein by mass spectrometry often does not allow its unique identification among possible proteins within a complete proteome, but determination of the masses of all fragments produced by digestion with trypsin almost always allows unique identification. Explain.

Short answer

Mass spectrometry of digestion fragments creates unique mass patterns, enabling precise protein identification.

Step-by-step solution

Introduction to Mass Spectrometry

Mass spectrometry (MS) is a technique used to determine the mass of molecules, including proteins. It measures the mass-to-charge ratio of ions to identify different substances within a sample.

Determination of Protein Mass

When the mass of a whole protein is determined by MS, it provides an overall mass that can match many proteins having similar masses due to the presence of many homologous proteins with similar sizes in large proteomes.

Fragmentation of Proteins

Proteins are often digested into smaller peptides by enzymes like trypsin. Trypsin cleaves proteins at specific sites, typically after lysine (K) or arginine (R), resulting in predictable fragments based on the protein's amino acid sequence.

Unique Identification by Peptide Mass Fingerprinting

By determining the mass of each peptide fragment produced by trypsin digestion using MS, a unique pattern or 'fingerprint' is obtained. This pattern corresponds to specific sequences of the original protein, reducing ambiguity.

Matching Patterns to Proteomes

Each unique fingerprint of peptide masses can be matched against databases of known protein sequences. If a complete match is found, the original protein is uniquely identified, even among thousands of proteins.

Conclusion

While whole protein mass can match multiple proteins, the combination of fragment masses provides a distinctive signature that allows for the unique identification within a proteome.

Key concepts

Protein Identification

Protein identification is a crucial step in understanding the functions and roles of proteins within biological systems. Proteins are composed of long chains of amino acids, and each sequence is unique to specific proteins.
Using mass spectrometry (MS), scientists can identify proteins based on their mass. However, when only the overall mass is known, many different proteins might match due to similarities, especially in large proteomes like those found in humans. This makes unique identification challenging using only the full protein mass.
Thus, to achieve accurate identification, additional steps like fragmentation and analysis of peptides are utilized, allowing for precise protein identification.

Trypsin Digestion

Trypsin digestion is a process used to fragment proteins into smaller peptides. Trypsin is an enzyme that cleaves peptide chains mainly at the carboxyl side of the amino acids lysine (K) or arginine (R). This specific cutting pattern leads to predictable and manageable protein fragments.
These smaller peptides are easier to analyze using mass spectrometry.
Trypsin digestion converts complex proteins into simpler pieces, providing more detailed insights into a protein’s structure by breaking it down into interpretable segments. This step is essential for applications like peptide mass fingerprinting, where the pattern of these fragments provides valuable information for protein identification.

Peptide Mass Fingerprinting

Peptide mass fingerprinting (PMF) is a method that uses the unique set of peptide masses obtained after trypsin digestion to identify proteins. After fragmentation, each peptide's mass is measured using mass spectrometry.
The result is a fingerprint that is distinctive for each protein because it reflects specific sequences in the original protein.
PMF involves matching these mass spectra against databases of known protein sequences. This comparison helps in pinpointing the exact protein, allowing for accurate and specific identification.

• Creates unique patterns of masses
• Reduces ambiguity in protein identification
• Effective in identifying proteins in complex mixtures

Proteome Analysis

Proteome analysis involves the study of the complete set of proteins produced by an organism. It's a huge field concerned with understanding how proteins function within cells, tissues, and entire organisms.
Mass spectrometry plays a central role in proteome analysis, especially when combined with techniques like trypsin digestion and peptide mass fingerprinting.
By fragmenting proteins and analyzing them, scientists can explore the vast diversity of the proteome, helping in discovering new proteins and understanding complex biological processes. This comprehensive approach is pivotal in fields such as drug discovery and disease research.