PepGenexShop our research products

Mass spectrometry (MS)

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of ions. An introductory review by Finehout and Lee describes it as an essential analytical tool in biological research that can characterize a wide variety of biomolecules, including sugars, proteins and oligonucleotides.[1]

How are peptides and proteins turned into ions?

Fenn and colleagues described electrospray ionization in 1989 as a technique for producing intact ions in vacuo from large and complex species in solution, which made mass spectrometric analysis applicable to large, fragile polar molecules.[2]

The same report states that electrospray spectra of large molecules show coherent sequences of peaks whose ions are multiply charged, each peak differing by one charge from its neighbors. The authors obtained spectra for biopolymers including oligonucleotides and proteins.[2]

In 1988 Karas and Hillenkamp reported laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.[3]

How is a peptide sequence read from mass spectra?

Hunt and colleagues described in 1986 a method for determining amino acid sequences by tandem mass spectrometry. The protein is degraded enzymatically or chemically to a collection of peptides, the peptides are fractionated by HPLC, and each fraction is analyzed by collision-activated dissociation mass spectrometry on a multianalyzer instrument.[4]

In that work, fractions containing as many as 10-15 peptides were analyzed directly, without further purification, and sequences were interpreted from the collision-activated dissociation spectra.[4]

Roepstorff and Fohlman proposed a common nomenclature for the sequence ions in mass spectra of peptides in 1984.[5]

How is mass spectrometry combined with liquid chromatography?

A review by Lian and colleagues describes liquid chromatography-mass spectrometry (LC-MS) as increasingly used to characterize synthetic peptides and their impurities, including structural isomers and stereoisomers, and proposes an LC-MS workflow for that characterization.[6]

In an LC-MS peptide map described by Mouchahoir and Schiel, the trace is a total ion chromatogram and each peak is mapped to peptides identified from the mass spectrometry data.[7]

ICH Q2(R2) gives an example validation for quantitative LC/MS of trace impurities, in which specificity may be inferred from specific and selective MS detection, such as an accurate m/z value, in combination with retention time.[8]

What does ICH Q6B list mass spectrometry for?

ICH Q6B lists mass spectrometry among the techniques for determining molecular size, alongside size exclusion chromatography and SDS-polyacrylamide gel electrophoresis. It also lists mass spectrometry among the techniques for identifying the fragments of a peptide map, alongside amino acid compositional analysis and N-terminal sequencing.[9]

The same guidance states that peptide mapping under reducing and nonreducing conditions, mass spectrometry or other appropriate techniques may be useful for determining the number and positions of free sulfhydryl groups and disulfide bridges. It lists mass spectroscopy among the methods by which deamidated, isomerized, oxidized and other modified forms may be detected and characterized.[9]

ICH Q2(R2) states that where the specificity of a technology can be ensured and predicted by technical parameters, such as the resolution of isotopes in mass spectrometry, additional experimental studies may not be required, if justified.[8]

What can a mass measurement not distinguish?

Isomers have an identical chemical formula and therefore the same mass-to-charge ratio, so identifying and specifically quantifying isomers in a complex matrix by mass spectrometry alone is not an easy task, as Rathahao-Paris and colleagues note.[10]

For synthetic peptides, Lian and colleagues place particular emphasis on structural isomers and stereoisomers, such as peptide epimers, among the impurities that LC-MS characterization has to address.[6]

ICH Q2(R2) recommends a combination of two or more procedures where one analytical procedure does not provide sufficient discrimination.[8]

Limitations

A mass match is not a measure of quantity. ICH Q2(R2) validates quantitative LC/MS separately, including its linearity, detection limit and quantitation limit.[8]

Identity and purity are separate specification items in ICH Q6B, so a mass consistent with the expected peptide does not by itself establish purity.[9]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Compound profiles whose identity section records the sequence and molecular formula that an identity result is compared against.

References

  1. Finehout EJ, Lee KH. An introduction to mass spectrometry applications in biological research. Biochem Mol Biol Educ. 2004;32(2):93-100. PMID 21706701 · DOI 10.1002/bmb.2004.494032020331
  2. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71. PMID 2675315 · DOI 10.1126/science.2675315
  3. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988;60(20):2299-2301. PMID 3239801 · DOI 10.1021/ac00171a028
  4. Hunt DF, Yates JR, Shabanowitz J, Winston S, Hauer CR. Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci U S A. 1986;83(17):6233-6237. PMID 3462691 · DOI 10.1073/pnas.83.17.6233 · PMC386476
  5. Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984;11(11):601. PMID 6525415 · DOI 10.1002/bms.1200111109
  6. Lian Z, Wang N, Tian Y, Huang L. Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography-Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives. J Am Soc Mass Spectrom. 2021;32(8):1852-1860. PMID 34110145 · DOI 10.1021/jasms.0c00479
  7. Mouchahoir T, Schiel JE. Development of an LC-MS/MS peptide mapping protocol for the NISTmAb. Anal Bioanal Chem. 2018;410(8):2111-2126. PMID 29411091 · DOI 10.1007/s00216-018-0848-6 · PMC5830484
  8. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
  9. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  10. Rathahao-Paris E, Abdoun S, Paris A, et al. Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix. J Mass Spectrom. 2024;59(5):e5026. PMID 38656572 · DOI 10.1002/jms.5026