Protein Protocols Protein Protocols

Nanoelectrospray MS/MS for Peptide Sequencing 705
As mentioned previously, tryptic peptides have the desirable feature that they contain
an N-terminal amino group and a C-terminal Lys or Arg residue, localizing protons at
both the N-terminus and the C-terminus of the peptide. Tandem mass spectra of tryptic
peptides very often contain a continuous y-ion series that can be readily assigned in the
m/z range above the doubly charged parent ion signal. The spectrum interpretation
strategy builds on this characteristic. It is guided by the demand to identify a protein in
sequence databases or to reliably sequence peptides for cloning of the cognate protein.
Several algorithms can identify proteins based on sequence database searches with
uninterpreted peptide tandem mass spectra (27,28). Such software tools are very useful
for a first screening of peptide tandem mass spectra because they immediately
identify peptides originating from known proteins. The Mascot search engine and a
number of other useful Internet-based services can be found via the URL http://
www.protein.sdu.dk.
2.9. Guidelines for Interpreting Tandem Mass Spectra
of Tryptic Peptides
The following list summarizes a few basic empirical rules that we use in interpreting
tandem mass spectra of tryptic peptides. Because peptides differ in their fragmentation
behavior in a sequence-dependent manner it is possible to find exceptions to these rules.
1. The goal of the interpretation is to find a series of peaks that belong to one ion series—for
tryptic peptides mostly y-ions (C-terminal fragments).
2. Initial peak selection: The high m/z region of a tandem mass spectrum is often straightforward
to interpret. Choose a large ion signal in this region as the “starting peak”.
3. Assembly of a partial amino acid sequence: Try to find ion signals that are precisely one
amino acid residue mass away from the starting peak (up or down in mass). We use software
which marks all the possibilities in the spectrum. This provides a good overview
whether there is more than one possibility for sequence assignments. If there is a repeating
pattern of fragment ion peaks with satellite peaks representing an H 2O loss (–18 Da) or an
NH 3 loss (–17 Da) a fragment ion series has been identified (for tryptic peptides a y–ion
series is more likely).
4. By repeating step 3 a peptide sequence tag consisting of two to four amino acids is
assembled that is subsequently used to identify a protein in the sequence database. As
default for tryptic peptides, the database is searched under the assumption that a y–ion
series was determined. However, even for tryptic peptides the tandem mass spectrum can
be dominated by b-ions if a peptide contained an internal basic residue or when the
C-terminal peptide of the protein had been sequenced.
2.10. Confirming Protein Identifications Made
via Peptide Tandem Mass Spectra
If a protein sequence is retrieved by a database search with a peptide sequence tag or
by similar methods then the amino acid sequence of the retrieved peptide should fit the
tandem mass spectrum in order to be called as a positive match. Two or more peptides
from a sample should independently identify the same protein in a database. To verify
a match, the peptide fragment masses must be correct within the expected error of the
mass measurement. For tryptic peptides the y-ion series should be nearly complete,
except when a peptide contains an internal proline residue (see below). The N-terminal