CHAPTER 5
De novo Sequencing of Proteins With Mass
Spectrometry Using the Differential Scanning Technique
M. WrLMI, G. NEUBAUER!, L. TAYLOR 2 , A. SHEVCHENKO!, A. BACHr l
1
Abstract
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-terminallabeling 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 % 18 0 labeling is the method of choice. In this
article a method is presented, called differential scanning, which addresses the
major limitations of methylation and 18 0 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 % 18 0 labelled fragment ions via their 1:1 16 0/ 18 0 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 y ions only tandem
MS spectrum and it can be exploited to improve computer algorithms to read out
the amino acid sequence automatically.
I European Molecular Biology Laboratory, Meyerhofstrasse I, D-69012 Heidelberg, Germany.
2 PE-Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8.
De novo Sequencing of Proteins With Mass
Spectrometry Using the Differential Scanning Technique
M. WrLMI, G. NEUBAUER!, L. TAYLOR 2 , A. SHEVCHENKO!, A. BACHr l
1
Abstract
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-terminallabeling 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 % 18 0 labeling is the method of choice. In this
article a method is presented, called differential scanning, which addresses the
major limitations of methylation and 18 0 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 % 18 0 labelled fragment ions via their 1:1 16 0/ 18 0 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 y ions only tandem
MS spectrum and it can be exploited to improve computer algorithms to read out
the amino acid sequence automatically.
I European Molecular Biology Laboratory, Meyerhofstrasse I, D-69012 Heidelberg, Germany.
2 PE-Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8.