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De Novo Peptide Sequencing and Amino Acid Analysis

A variety of methods exist to determine the sequence or amino acid composition of a peptide. Typically, the peptide is purified using reverse phase HPLC, SDS-PAGE, or PVDF blotting prior to analysis. Methods for analysis include:

  • Edman degradation (N-terminal sequencing) - (N-TS) - A technique invented by Pehr Victor Edman. The sequence is determined by the sequential treatment of the peptide with phenylisothiocyanate (PITC) which results in the cleavage and derivatization of the N-terminal amino acid. The resulting amino acids are analyzed in the order they are cleaved and the original peptide sequence is deconvoluted.

  • C terminal sequencing - (C-TS) - Peptide degradation can be accomplished chemically (the C-terminal carboxyl group is activated as a mixed anhydride and then reacted with isothiocyanate) or enzymatically (using one of several carboxypeptidase enzymes). This method typically requires larger amounts of sample versus N-terminal sequencing.

  • internal sequencing - (I-S) - This method is used when the N-terminus or C-terminus is blocked. Alternatively, it can be used if it is not known whether or not the termini are blocked, and the user does not want to take a chance on having a problem

  • mass spectrometry - (MS) - de novo peptide sequencing can also be performed using mass spectrometry instrumentation

  • amino acid analysis - (AAA) - Peptides are cleaved to their individual amino acids under acidic conditions (e.g., 6 N HCl). The resulting mixture is then quantified to determine the percentage of each amino acid in the original peptide.

Here is a list of companies with a brief description of the kind of services that they provide:

  1. 21st Century Biochemicals - (MS) -  LC-MS/MS for mass determination, peptide mapping, and peptide sequencing, including the identification of protein phosphorylation sites

  2. AAIPharma - (MS) - trypsin peptide mapping; protein sequencing; and characterization of disulfide bonds, protein oxidation, end capping, glycosylation sites, phosphorylation sites, and other post-translational modifications using LC-MS/MS

  3. Alberta Peptide Institute - (AAA, MS) - quantitative and qualitative amino acid analysis and peptide/protein sequencing from electroblotted samples.  Additional services include in-gel protein identification (LC-MS/MS) and protein MW determination (MALDI-TOF)

  4. Alta Bioscience - (N-TS, AAA) - under the ISO/IEC 17025 quality standard.  They also perform peptide purity analyses and MALDI-TOF molecular weight determinations of biomolecules.

  5.  AIBioTech - (N-TS, C-TS, AAA, MS)

  6. Anaspec - peptide standards for sequencing analysis

  7. Biosynthesis - (N-TS, AAA, MS) - amino acid analysis and peptide/protein sequencing

  8. Cambridge Peptides - (N-TS) - Edman degradation to GLP and cGMP standards

  9. Eurosequence - (N-TS, C-TS, I-S, AAA) - amino acid analysis, peptide/protein sequencing using microsequecing techniques and mass spectrometry. They also offer a GMP amino acid sequencing service which is used for qualification testing during GMP manufacturing.

  10. Harvard University Microchemistry and Proteomics Analysis Facility - (N-TS, C-TS, AAA, MS) - peptide/protein ID using microcapillary nano LC-MS/MS and MALDI/TOF

  11. Leeds Protein Sequencing Facility - (N-TS, MS)

  12. Proteomics and Metabolomics Facility - Colorado State University - (MS) - MS/MS spectra are collected using MALDI-TOF/TOF for de novo sequencing of peptides and the identification of post-translational modifications

  13. Midwest Bio Services - (MS) - protein identification by tandem mass spectrometry using nano-LC/MS/MS with complete sample processing.  They also provide the identification of proteins in gel slices which includes sample preparation, digestion, peptide extraction, nano-LC/MS/MS data acquisition, and SEQUEST data analysis.

  14. M-Scan - (N-TS, C-TS, AAA, MS) - With locations in Europe, the United States, and Asia, they are a FDA inspected contract research laboratory that is GLP/cGMP compliant

  15. The Protein Facility of the Iowa State University Office of Biotechnology - (N-TS, MS)

  16. Proteome Factory - (N-TS, MS) - contract research and fee-for-services that include protein identification, sequencing, quantification, and proteomics

  17. Proteomics International - (N-TS, MS) - including the MS analysis of intact proteins and peptides

  18. ProtTech - (MS) - peptide/protein ID by nano LC-MS/MS sequencing

  19. Rocky Mountain Instrumental Laboratories - (MS)

  20. SSCI - (N-TS, AAA, MS) - specializing in pre-formulation studies under cGMP conditions including N-terminal sequence analysis, amino acid analysis, MALDI-TOF mass spec, peptide mapping, peptide mass fingerprinting, spectroscopy and aggregation studies.

  21. W.M. Keck Biotechnology Resource Laboratory - (N-TS, AAA, MS)

  22. Vimta Labs - (MS) - 2D Gel,  MALDI TOF TOF for intact mass analysis, peptide mass fingerprinting, MS/MS peptide sequencing
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