De novo peptide sequencing is performed without prior knowledge of the amino acid sequence. It uses computational approaches to deduce the peptide sequence directly from mass spectrometry spectra. The main principle is to use mass differences between fragment ions like y and b ions to calculate amino acid residues. While it can identify previously unknown sequences, there is uncertainty in the complete sequence and difficulty determining directionality sometimes. Creative Proteomics offers de novo sequencing services using high-performance mass spectrometry and computational algorithms.

1. De Novo Peptide Sequencing
Creative Proteomics Presentation

2. De Novo Peptide Sequencing: peptide sequencing performed
without prior knowledge of the amino acid sequence
De novo: Latin for “over again” or
“anew”
De Novo Peptide Sequencing

3. Derives the peptide
sequences without a
protein database
It uses computational
approaches to deduce the
sequence of peptide directly
from the spectra.
For un-sequenced
organisms, antibodies,
peptide with PTMs, and
endogenous peptides
Introduction

4.  3 types of backbone bonds can be broken to
form peptide fragments: alkyl carbonyl (CHR-
CO), peptide amide bond (CO-NH), and amino
alkyl bond (NH-CHR)
 6 types of fragmentation ions: the N-terminal
charged fragment ions are classed as a, b or c;
the C-terminal charged ones are classed as x,
y or z
 Because the peptide amide bone (CO-NH) is
the most vulnerable, the most common peptide
fragments observed in low energy collisions
are a, b and y ions
Peptide fragmentation
Types of fragmentation ions
Figure 1. Different types of fragmentation ions
Ma B, Johnson R. De novo sequencing and homology searching[J]. Molecular & cellular proteomics, 2012, 11(2): O111. 014902.

5.  Collision induced dissociation (CID)
Peptide fragmentation
Methods for peptide fragmentation
 Electron capture dissociation (ETD ) and Electron transfer dissociation
(ECD)
• Also known as collisionally activated dissociation(CAD)
• The most common form of fragmentation
• Ions obtain high kinetic energy and collide with neutral molecules. Some
of the kinetic energy is converted into internal energy which leads to
bond breakage and the fragmentation of the molecules into smaller
fragments
• Result in the formation of b and y series ions from the precursor ion
• Have been implemented in the recent mass spectrometer
• Ions are fragmented after reaction with electrons.
• Form c and z type ions through cleavage of the peptide bond between
the amino group and alpha carbon
Figure 2 Fragment ion types produced following either CAD or ETD
Coon J J. Collisions or electrons? Protein sequence analysis in the 21st
century. 2009.

6. The mass can usually uniquely determine the residue. The main principle of de novo sequencing is to use the mass
difference between two fragment ions to calculate the mass of an amino acid residue on the peptide backbone.
Principle
For example, the mass difference between the y7 and y6 ions in the following figure is equal to 101, which is the mass
of residue T.

7. Principle
 Thus, if one can identify either the y-ion or b-ion series in the spectrum, the peptide sequence can be
determined. However, the spectrum obtained from the mass spectrometry instrument does not tell the ion
types of the peaks, which need either an expert or a computer algorithm to figure out.
 There are couples of software packages used for de novo sequencing, such as PEAKS, Lutefisk,
PepNovo, SHERENGA and so on.
 Notes: y and b ion fragments which contain the amino acid residues R, K, Q, and N may appear to lose
ammonia(-17). Y and b ion fragments which contain the amino acid residues S, T, and E may appear to
lose water(-18).

8. Advantages
 Identify previous unknown
peptide sequences
 Search for posttranslation
modifications or for the
identification of mutations by
homology based software
Disadvantages
 Uncertainty regarding the
complete peptide sequence.
 Sometimes it can be difficult
to determine the
directionality of a sequence
 Low mass accuracy fragment
ion measurements cannot
distinguish between lysine
and glutamine (differ by
0.036 Da) nor between
phenylalanine and oxidized
methionine (differ by 0.033
Da).
Advantages and Disadvantages

9. At Creative Proteomics
Equipped with state-of-the-art technologies
such as high-performance liquid
chromatograph coupled to tandem mass
spectrometry (LC-MS/MS) and advanced
computational algorithms, Creative Proteomics
can offer accurate and fast de novo
peptide/protein sequencing service customized
to your needs.
Our Services
De Novo Peptides/Proteins
Sequencing Service

10. Thank you
Please contact us for more information
Web
Email
www.creative-proteomics.com
info@creative-proteomics.com