PROTEIN SEQUENCING 1 methods for protein sequencing Edman Degradation is one of the N-terminal amino acid sequence analysis methods for peptide chains/proteins sequencing. The protein is reacted with PITC under weakly basic conditions, and then treated with an acid to free the amino terminal residue of the peptide chain in the form of PTH-AA for subse- quent analysis. Peptide Mapping analysis is an effective method for rapidly localizing protein sequences and is a commonly used strategy in protein identification.The method uses mass spectrometry for peptide analysis, and compares the obtained spectra with a protein database to obtain amino acid information. De Novo Protein Sequencing is a method based on the enzymatically cleaved peptides that exhibit regular fragmentation in mass spectrometry to obtain amino acid information from the mass differences in regular mass spectral peak. 2 the progress of these three methods Protein N' Labeling Purified protein Purified Protein PITC (Trypsin, Lys-C, Glu-C, Protein digestion chymotrypsin, Asp-N Protein digestion Release and Arg-C) PITC Labeling Peptide mixture Peptide Mixture PTH PITC Separation and Separation and Release Purification by HPLC Purification by HPLC PTH-AA PITC analysis by HPLC Target Peptides PITC Next cycle of Target peptides PITC reaction MALDI-MS/MS MALDI/ESI-CID/HCD/ETD y3 PTH y2 b3 b2 Intensity Intensity Intensity Intensity PTH-AA m/z m/z m/z m/z analysis by HPLC Compare the peptide masses Using the de novo sequencing with protein databases analysis software to analyze the peak map of specific fragmentation ions to obtain the mass of amino acids 3 characteristics (1) Identify the exact N-termi- (1) High specific enzyme diges- (1) Does not require a refer- nal amino acid. tion produces peptide frag- ence database to deduce (2) The released amino acids ments ranging in size from 400 full-length or partial tag-based are identified and quantifed by < m/z < 5000, corresponding to peptide sequences directly chromatography. ~4–45 amino acid residues from experimental tandem (3) Enable N- terminal sequenc- with sufficient specificity, mass spectrometry spectra. ing of proteins in mixtures. consistent with the scanning (2) Identify novel peptides, range of common mass analyz- unsequenced organisms and ers. antibody drugs. (1) It will not work if the N-ter- (2) Combination of specific minus has been chemically and non-specific protein Sample contamination, modified. enzymes to achieve 100% isotope peak interference, loss (2) Sequencing wil stop if a coverage of any protein (poly- of important b-ion peak or non-α-amino acid is encoun- peptide). y-ion peak in most maps tered. caused by incomplete dissocia- (3) Larger proteins cannot be tion of peptide segments, loss sequenced by the Edman (1) Only peptide mass are mea- of information at n-terminal sequencing. sured, contamination from and c-terminal as well as vari- (4) Edman degradation is gen- other protein can be interfer- ous noise interference will erally not useful to determine ence to the accuracy of lead to decreased accuracy of the positions of disulide bridg- protein analysis. ab initio sequencing method es. (2) Only protein from the data- base can be identified. and difficult to obtain correct peptide sequence information. 4 applications This study presents an improvement of ADC This study combines standard proteogenomics peptide mapping protocol to characterize the with peptide de novo sequencing to refine anno- drug-loaded peptides by LC-MS analysis. All the tation of the well-studied model fungus Sordaria steps of this protocol including enzymatic diges- macrospora. tion were improved to maintain the hydrophobic drug-loaded peptides in solution by the addition of solvents. This is the first time that the payload positional 104 so-far hidden proteins and annotation chang- isomers on the heavy chain of an ADC conjugated es in 575 genes, including 389 splice site refine- on native cysteines, were characterized by ments were detected. peptide mapping LC MS analysis. This approach provides peptide-level evidence for This method can provide important structural 113 single-amino-acid variations and 15 C-terminal information about the locations of conjugation protein elongations originating from A-to-I RNA sites on cysteines residues of heterogeneous editing. ADCs. Janin-Bussat, Marie-Claire, et al. "Characterization of Blank-Landeshammer, B., et al. "Combination of antibody drug conjugate positional isomers at cyste- Proteogenomics with Peptide De Novo Sequencing ine residues by peptide mapping LC–MS analysis." Identifies New Genes and Hidden Posttranscription- Journal of Chromatography B 981 (2015): 9-13. al Modifications." mBio 10.5 (2019): e02367-19. Email: [email protected] Tel: 1-631-275-3058 © 2008-2019 Creative Proteomics. All rights reserved..