De novo sequencing of proteins with mass spectrometry using the differential scanning technique

Protein identification in complete sequence databases or partial sequence databases is done preferentially by mass spectrometric techniques. However, protein de novo sequencing with mass spectrometry is more difficult to achieve. Generally, it is done by C-terminal labeling of the peptide. C-terminal fragment ions are identified in the tandem mass spectrum by the additional mass of the label. This very often allows to assign the correct amino acid sequence to a fragment spectrum of a tryptic peptide. When sub-isotopically resolved tandem mass spectra generated with a triple quadrupole machine are to be interpreted, methylation is the preferred labeling method since the mass shift is at least 14 Da. When working with a quadrupole time of flight machine which generates isotopically resolved fragment spectra, 50 % O-18 labeling is the method of choice. In this article a method is presented, called differential scanning, which addresses the major limitations of methylation and O-18 labeling preserving at the same time the sensitivity of the analysis. For the triple quadrupole based de novo sequencing there is the need to do two separate tandem MS investigations, one for the unmodified peptide mixture and the second for the methylated one. For the quadrupole time of flight based technique the difficulty is to clearly and unambiguously identify the 50 % O-18 labelled fragment ions via their 1:1 O-16/O-18 isotopic cluster in case of overlap with chemical noise ions and other ions mimicking the characteristic isotopic distribution. When the differential scanning technique is used additional information is generated to identify the C- terminal fragment ions. This information can be used to generate a simplified gamma ions only tandem MS spectrum and it can be exploited to improve computer algorithms to read out the amino acid sequence automatically.