De novo peptide sequencing-creative proteomics

The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence. It uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra.This method can obtain the peptide sequences without a protein database. It can be used for un-sequenced organisms, antibodies, peptides with posttranslational modifications, and endogenous peptides.

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. S ome 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 y 7  and y 6  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 [email protected]