Protein Protocols Protein Protocols

706 Jensen and Wilm
b 2 and a 2 fragment ions, generated at relatively high collision energy, should be present
in the low m/z region. Odd fragmentation patterns should reflect the amino acid
sequence as discussed in the following paragraphs.
Peptides that contain internal basic residues (lysine or arginine) do not fragment in
the vicinity of these residues because a charge is localized at the sidechain of the basic
residue and therefore not available for amide backbone cleavage. If the triply charged
precursor ion was fragmented then doubly charged y ions are present in the spectrum.
Internal proline residues deserve special attention. Cleavage of the C-terminal bond
of a proline is observed to a low degree. The N-terminal bond of a proline is labile
giving rise to an intense y–ion fragment. Internal fragmentation of peptides containing
a proline often confirm a sequence: The y–ion generated by fragmentation at the
N-terminal side of Pro will dissociate a second time to produce a y–ion series which is
superimposed on the original y–ion series. However, the b–ions generated from this
Pro containing fragment serve to confirm the C-terminal part of a peptide sequence up
to and including the internal proline residue.
Isoleucine and leucine cannot be differentiated by amide bond cleavage alone
because they have the same elemental composition and therefore identical molecular
weight. Pairs of amino acid with identical nominal mass, Lys/Gln (128 Da) and oxidized
Met/Phe (147 Da), can often be distinguished. Lys and Gln differ in their basicity and
trypsin cleaves C-terminal to Lys and not at Gln so internal Lys is rarely found in
tryptic peptides. However, if the latter is the case then the tryptic peptide usually acquire
an additional proton for a total of three charges and the tandem mass spectrum will
often contain a doubly charged y-ion series. If an internal Lys residue is suspected then
the peptide mixture can be inspected for the presence of the limit peptide produced by
tryptic cleavage at this Lys residue. Oxidized methionine (147.02 Da) and phenylalanine
(147.07 Da) residues are differentiated relatively easily as follows. Tandem mass
spectra of peptides which contain an oxidized methionine residue (i.e., methionine sulfoxide)
display satellite peaks that appear 64 Da below each methionine sulfoxidecontaining
y-ion fragment due to loss of CH 3SOH from methionine sulfoxide (29,30).
The oxidation reaction is often not complete so inspection of the peptide mass spectrum
(Q 1 spectrum) may reveal a peptide 16 Da below the one containing oxidized
methionine. The high resolution and mass accuracy of Q-TOF type instruments enable
differentiation of Gln/Lys and OxMet/Phe by accurate mass determination.
If no proteins are retrieved with a simple database search then redo the search under
the assumption that some of the amino acids in the peptide are modified, containing for
example, an oxidized methionine or a S-acrylamidocysteine. An error tolerant search
can be launched in which only partial correspondence between the peptide sequence
tag and a database entry is required (25). Additional information about the protein can
be used to select possible candidates if more than one protein sequence is retrieved
(such as protein size, organism, or function).
If a protein cannot be identified by any of its peptide sequence tags we conclude that
it is unknown. For proteins from human, mouse, or other model organisms, the peptide
sequence tags are then screened against a database of expressed sequence tags (ESTs)
(31). ESTs are short stretches of cDNA, that is, single-stranded DNA generated from
expressed mRNA by reverse transcriptase, and thus represent the set of expressed genes
in a given cell line. If a database search retrieves a cDNA sequence then library screen-