Peptide Identification & Advanced Mass Spectrometry Analysis
Mass spectrometry (MS) provides molecular weight confirmation, structural insight, and identity support for peptide materials. When combined with chromatographic separation, MS enables high-confidence evaluation of peptide integrity, impurity species, and modification states.
Why Mass Spectrometry Is Critical for Peptide Programs
- Accurate molecular weight confirmation
- Verification of expected modifications (oxidation, phosphorylation, labeling, etc.)
- Impurity clarification and peak assignment
- Orthogonal confirmation alongside HPLC purity analysis
Ionization Techniques
MALDI-TOF (Matrix-Assisted Laser Desorption Ionization – Time of Flight)
MALDI-TOF is commonly used for rapid intact mass confirmation. The peptide is co-crystallized with a matrix and ionized via laser excitation. Time-of-flight detection measures mass-to-charge ratio (m/z), typically generating singly charged species suitable for fast molecular weight assessment.
- Rapid identity confirmation
- High throughput screening
- Effective for relatively pure peptide samples
LC-ESI-MS (Liquid Chromatography – Electrospray Ionization MS)
LC-MS using Electrospray Ionization (ESI) allows peptides to enter the gas phase directly from solution. ESI typically produces multiply charged ions, enabling analysis of larger peptides with high sensitivity.
- Chromatographic separation prior to MS detection
- Improved impurity resolution
- Charge state distribution analysis
Charge States & Deconvolution
ESI-generated peptides often appear as multiple charge states. Deconvolution algorithms are applied to convert the m/z envelope into a single neutral molecular mass for direct comparison with theoretical mass values.
High-Resolution Mass Spectrometry
High-resolution MS improves mass accuracy and enhances the ability to distinguish closely related species. Accurate mass measurement strengthens confidence in identity confirmation and impurity differentiation.
MS/MS (Tandem Mass Spectrometry)
When deeper structural confirmation is required, tandem MS (MS/MS) may be used. Selected precursor ions are fragmented to generate sequence-informative fragment ions. This supports confirmation of amino acid composition and specific modification sites (scope-dependent).
Common Observations in Peptide MS
- Main molecular ion corresponding to theoretical peptide mass
- Oxidation (+16 Da shifts)
- Adduct formation (e.g., sodium or potassium adducts)
- Deletion or truncation species
- Modification-related mass shifts
Interpretation Considerations
Observed mass values are compared against calculated theoretical mass derived from sequence and modification data. Adducts, salt interactions, and ionization behavior are evaluated during spectral interpretation.
Mass spectrometry provides molecular-level evidence for peptide identity. Complete structural confirmation may require integration with chromatographic purity data and additional orthogonal techniques when appropriate.
Reporting
Reports include annotated spectra, observed and theoretical mass comparison, charge state distribution (when applicable), and interpretive commentary. LC-MS reports may include chromatographic traces with peak-specific mass assignments.
Bio-Synthesis, Inc. provides peptide identification and advanced mass spectrometry analysis aligned with GLP / ISO 13485 quality systems for regulated non-GMP peptide programs.