Protein Sequencing and Identification by Mass Spectrometry.pptVIP

Protein Sequencing and Identification by Mass Spectrometry Masses of Amino Acid Residues Protein Backbone Peptide Fragmentation Peptides tend to fragment along the backbone. Fragments can also loose neutral chemical groups like NH3 and H2O. Breaking Protein into Peptides and Peptides into Fragment Ions Proteases, e.g. trypsin, break protein into peptides. A Tandem Mass Spectrometer further breaks the peptides down into fragment ions and measures the mass of each piece. Mass Spectrometer accelerates the fragmented ions; heavier ions accelerate slower than lighter ones. Mass Spectrometer measure mass/charge ratio of an ion. N- and C-terminal Peptides Terminal peptides and ion types N- and C-terminal Peptides N- and C-terminal Peptides N- and C-terminal Peptides N- and C-terminal Peptides Peptide Fragmentation Mass Spectra The peaks in the mass spectrum: Prefix Fragments with neutral losses (-H2O, -NH3) Noise and missing peaks. Protein Identification with MS/MS Tandem Mass-Spectrometry Breaking Proteins into Peptides Mass Spectrometry Tandem Mass Spectrometry Protein Identification by Tandem Mass Spectrometry Tandem Mass Spectrum Tandem Mass Spectrometry (MS/MS): mainly generates partial N- and C-terminal peptides Spectrum consists of different ion types because peptides can be broken in several places. Chemical noise often complicates the spectrum. Represented in 2-D: mass/charge axis vs. intensity axis De Novo vs. Database Search De Novo vs. Database Search: A Paradox The database of all peptides is huge ≈ O(20n) . The database of all known peptides is much smaller ≈ O(108). However, de novo algorithms can be much faster, even though their search space is much larger! A database search scans all peptides in the database of all known peptides search space to find best one. De novo eliminates the need to scan database of all peptides by modeling the problem as a graph search. De novo Peptide Sequencing  Theoretical Spectrum Theoretical Spectrum (cont’d) Theo