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.
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
Editor's Notes
Hello, welcome to watch Creative Proteomics’Video. As we know, through tandem mass spectrometry is one of the most powerful tool for protein identification. Today, we are going to learn some basic knowledge about de novo peptide sequencing.
De novo is Latin which means “over again” or “anew”. The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence.
This method can obtain the peptide sequences without a protein database, which can overcome the limitations of database-dependent methods like peptide mass fingerprinting. It can be used for un-sequencd organisms, antibodies, peptide with posttranslational modifications (PTMs), and endogenous peptides. In addition, it uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra.
In a tandem mass spectrometer, the peptide is fragmented along the peptide backbone and the resulting fragment ions are measured to produce spectrum. There are 3 ways to break bonds to form peptide fragment: alkyl carbonyl (CHR-CO), peptide amide bond (CO-NH), and amino alkyl bond (NH-CHR) Therefore, it can form 6 types of fragmentation ions, including the N-terminal charged fragment ions which are classed as a, b or c, and the C-terminal charged ones which are classed as x, y or z. And 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
De novo methods use the knowledge of the fragmentation methods employed in the MS, such as CID, or ECD. CID, Collision induced dissociation, also known as collisionally activated dissociation, is the most common form of fragmentation. In this method, The ions can 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. This method results in the formation of b and y series ions from the precursor ion. The Electron capture dissociation, ETD and Electron transfer dissociation, ECD, have been implemented in the recent mass spectrometer. In these methods, Ions are fragmented after reaction with electrons. After fragmentation, it Forms c and z type ions through cleavage of the peptide bond between the amino group and alpha carbon.
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. 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.
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 require either a human expert or a computer algorithm to figure out during the process of de novo sequencing. There are couples of software packages used for de novo sequencing, such as PEAKS, Lutefisk, PepNovo, SHERENGA and so on.But there are some notes you have to mind. y and b ion fragments which contain the amino acid residues R,K,Q, and N may appear to loose ammonia(-17). y and b ion fragments which contain the amino acid residues S, T, and E may appear to lose water(-18).
De novo sequencing can identify previous unknown peptide sequences. In addition, it can search for posttranslational modifications or for identifications of mutations by homology based software. however, de novo sequencing will not be able to derive a complete sequence or will have uncertainty in a portion of the derived sequence. And sometimes it can be difficult to determine the directionality of a sequence. Low mass accuracy fragment ion measurements cannot distinguish between lysine and glutamine which differ by 0.036 Da nor between phenylalanine and oxidized methionine which differ by 0.033 Da.
At Creative Proteomics, we can provide de novo peptides or protein service. 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, we can offer accurate and fast de novo peptide/protein sequencing service customized to your needs.
Thanks for watching our video. At creative proteomics, we provide the most reliable services for protein identification not only de novo peptide sequencing, but some other technologies. If you have any questions or specific requirements. Please do not hesitate to contact us. We are very glad to cooperate with you.