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De novo protein sequence analysis
De novo protein sequence analysis





de novo protein sequence analysis
  1. #DE NOVO PROTEIN SEQUENCE ANALYSIS FULL#
  2. #DE NOVO PROTEIN SEQUENCE ANALYSIS SERIES#

The “depth of coverage” for an amino acid is defined as the number of unique PSMs covering the amino acid. The second and third columns list the median depths of coverage of the HCDR3 and the other variable portions, respectively. The first column lists the protease combination, where each protease is represented by a single letter code: P (Pepsin), T (Trypsin), C (Chymotrypsin), A (Asp-N), and L (Lys-C). The median depth of coverage for the HCDR3 region and the remaining portion of the variable regions are listed in the following table for each combination of the proteases used in this study. Sequence motif analysis was performed using SeqtoLogo. Yeast complex samples were analyzed with Search GUI-3.3.13 and PeptideShaker-1.16.37 using no enzyme restriction, 25ppm with protein, peptide and 1% psm FDR, while considering a combination of X! tandem, MS-GF+ and Comet. Data analysis:ĭe novo peptide sequencing was performed with Novor search algorithm and the protein sequences were assembled with REmAb ® ( Figure 1). ETD spectra were acquired with three different collisions energies.

#DE NOVO PROTEIN SEQUENCE ANALYSIS SERIES#

Protein lysates were analyzed with an Orbitrap Fusion™ Series Tribid™ instrument (ThermoFisher Scientific, CA, US) coupled to the LC Evosep One (Evosep, Denmark) in both HCD and ETD mode. department of Commerce), monoclonal mAb04 and yeast extracts (Promega, WI, US). Trypsin, Lys-C, Chymotrypsin, Pepsin, A/P, and recombinant Asp-N proteases Promega (Promega, WI, US) were used to digest NISTmAb humanized IgG1 monoclonal antibody RM8671 (National Institute of Standards and technology, U.S.

#DE NOVO PROTEIN SEQUENCE ANALYSIS FULL#

  • REmAb ®’s de novo protein sequencing established protocol includes orthogonal and protease cocktails optimized to delivery highly accurate and full coverage protein sequences.
  • Other orthogonal approaches also contribute to increase in accuracy for de novo protein sequencing.
  • A combination of proteases can maximize coverage during LC-MS/MS-based sequencing.
  • sequences to increase antibody sequencing accuracy. The assembled mAbs demonstrate that a combination of existing proteases with orthogonal activities significantly increases confidence scores in de novo protein sequencing however, there is a need for new proteases targeting specific amino acid(s) (a.a.) or a.a. Protein sequences were assembled using REmAb ®. De novo peptide sequencing was performed with Novor search algorithm. The new Pro/Ala (A/P) protease was tested to characterize NIST and MAB04HC antibody standards. Each antibody protein sample was digested separately with different proteases and analyzed by LC-MS/MS. MS data for 166 monoclonal antibodies were compiled for use in this study. In this study, we conducted a large-scale statistical analysis of protein sequencing data from samples digested with multiple proteases to understand the impact of using different combinations of proteases to improve the depth of sequence coverage in the application of de novo protein sequencing.
  • SPR Antibody-Antigen Interaction Analysis.






  • De novo protein sequence analysis