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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2006; 20: 933–936
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2382
To the Editor-in-Chief
Sir,
Distinctive mass losses of tryptic
peptides generatedbymatrix-assisted
laser desorption/ionization time-of-
flight/time-of-flight
Protein identification by mass spectro-
metry can be performed by peptide
mass fingerprinting and/or peptide
sequencing by tandem mass spectro-
metry (MS/MS).1 Sequence-specific
fragment ion spectra are dominated
by ions resulting from cleavage at the
amide bonds. According to the frag-
ment ion nomenclature of Roepstorff
and Fohlmann2 and Biemann,3 a, b,
b-H2O, b-NH3, y, y-H2O, and y-NH3
are frequently observed in MS/MS
spectra, usually dominated by b- and
y-ion series.4 However, the automatic
interpretation of MS/MS spectra by
computer algorithms is still frequently
ineffective and manual intervention is
often necessary.5 Furthermore, differ-
ent instrumentation for mass spectro-
metry can result in different features of
the MS/MS spectra. High-throughput
analysis with high sensitivity using
matrix-assisted laser desorption/ioni-
zation time-of-flight/time-of-flight
(MALDI-TOF/TOF) instruments is
well suited for proteome analyses.6 In
particular, MALDI-TOF/TOF can be
used successfully for peptide sequen-
cing by collision-induced dissociation
(CID) and post-source decay (PSD).
The most intense fragment ions gener-
ated by MALDI-MS are generally due
to fragmentation on the carboxy-term-
inal side of aspartic acids. Further-
more, proline residues produce
intense ions because of fragmentation
on the amino-terminal side of proline.
However, we observed mass peaks in
the high-mass range of fragmentation
spectra generated by MALDI-TOF/
TOF which did not correspond, with
one exception, to the usual ion series
(a, b, b-H2O, b-NH3, y, y-H2O, y-NH3)
(Table 1). Therefore, we analyzed some
thousand fragmentation mass spectra
generated during proteome analyses of
human Jurkat T cells, Mycobacterium
tuberculosis H37Rv, and Salmonella
enterica subspecies typhimurium
SL1344 to identifymass losses of tryptic
Copyright # 2005 John Wiley & Sons, Ltd.
RCM
Letter to the Editor
1825 1855 1885 1915 1945 1975Mass (m/z)
880 910 940 970 1000 1030Mass (m/z)
1225 1255 1285 1315 1345 1375Mass (m/z)
102030405060708090
100
ytisnet
nI%
102030405060708090
100
ytisnet
nI%
102030405060708090
100
ytisnet
nI%
A
B
C
86.2631
09.8131
18.4321
61 .0591
21.59 81
9 0.538 1
93.4101
45.0 59
84.99 8
Figure 1. Examples of distinct mass losses of tryptic
peptides. The tryptic peptide of human heterogeneous
nuclear ribonucleoprotein (hnRNP) L with the sequence
451NNRFSTPEQAAK462 showed the mass losses of 44
and 128Da compared to the parent mass. These mass
losses could be assigned to the threonine and the C-
terminal lysine with internal arginine (A). The peptide
279DYDDMSPR286 of human hnRNP K displayed a mass
loss of 64Da due to oxidized methionine with higher
intensity than the parent mass and a mass loss of 115Da
because of the N-terminal aspartic acid (B). Cysteine
modified by propionamide and N-terminal aspartic acid
were observed for the tryptic peptide 46DAGYEF-
DICFTSVQKR61 of human phosphoglycerate mutase 1
due to the mass losses of 55 and 115Da (C). Notably,
within this spectrum, the mass loss characteristic for
threonine was not clearly detected.
peptides. Beside the well-known neu-
tral loss of phosphorylated peptides
and oxidized methionine-containing
peptides, we observed the loss of 14Da
of methylated aspartic/glutamic acid-
containing peptides. Furthermore, the
occurrence of internal arginines, threo-
nines, and cysteines modified by pro-
pionamide can be predicted. Yn� 1 ions
were only observed for N-terminal
aspartic acid but no other amino acid.
An overview and explanation of the
distinctive mass losses of tryptic pep-
tides generated by MALDI-TOF/TOF
is shown in Table 1.
Internal arginines were clearly
detected as bn� 1þH2O ions (Table 1).
Amass loss of 156Dawasdetected if an
argininewas at theC-terminal end, and
a mass loss of 128Da (Fig. 1(A)) if a
lysine was located at the C-terminal
end of peptides containing internal
arginines. This was already considered
in the MS/MS rules for the Chem-
Score.7 However, characteristic mass
losses of internal lysines, the second
most basic amino acid after arginine,
were not found (Figs. 1(C) and 2).
The mass shift of 115Da could be
related to the yn� 1 ion of N-terminal
aspartic acid (Figs. 1(B) and 1(C)). No
further yn� 1 ions of all other less acidic
amino acids at the N-terminal end of
peptides were found.
The mass loss of 64Da of the parent
mass could easily be assigned to single
oxidized methionine (Fig. 1(B)). How-
ever, the mass shift of 64Da due to the
loss of methanesulfenic acid (CH3SOH)
was only observed for the parent
mass,8 whereas electrospray ionization
(ESI)-MS/MS of peptides with oxi-
dized methionine usually results in
pairs of mass peaks with a difference
of 64Da.9,10
A mass shift of 55Da could be
assigned to cysteines modified by
propionamide, a common modifica-
tion that occurs during gel electrophor-
esis (Fig. 1(C)).10,11 The only putative
explanation for this mass shift under
consideration of the present atoms was
the loss of vinylimine under generation
of cysteine sulfenic acid. Interestingly,
we observed frequently the immonium
ion of themodified cysteine at 147.0Da
within these mass spectra, a mass
which could be confused with the y1ion of C-terminal lysine at 147.1Da. In
contrast, the neutral loss of propiona-
mide (71Da) was observed by CID
using ion trap ESI-MS/MS only with
the singly charged ion but not with the
doubly charged ion.10
The only common feature of the
mass shift of 44Da compared to the
parent mass was the fact that all identi-
fied peptides contained a threonine
100 420 740 1060 1380 1700Mass (m/z)
100 420 740 1060 1380 1700Mass (m/z)
A
B
102030405060708090
100
ytisnet
nI%
102030405060708090
ytisnet
nI%
100
27.706137.1261
26 .7061
y1 y2
y5
y6
y10y7 y8y9 y11
y1 y2
y5 y6
y12y3 y4
y10y7
y12
y4
Figure 2. MS/MS spectra of an N-terminal acetylated tryptic
peptide and its methylated protein species. The MS/MS
spectrum of the N-terminal acetylated tryptic peptide of human
chromatin assembly factor 1 subunit C with the sequence
1ADKEAAFDDAVEER14 and m/z 1607.72Da is shown in (A).
The MS/MS spectrum of the methylated peptide with m/z of
1621.73Da is depicted in (B). Y ions according to the non-
methylated peptide are indicated in both spectra. The intensity
of the non-methylated in comparison to the neighbouring
methylated y ions decreased with increasing number of
aspartic and glutamic acids. Therefore, it can be concluded
that a mixture of the mono-methylated peptide modified at
different sites was fragmented. Please note that no hint as to
the acetylation of the peptide was observed.
Table 1. Overview of the distinctive mass losses of tryptic peptides generated by
MALDI-MS/MS
Mass shift (Da) Intensity* Ion Legend
156 � bn� 1þH2O Internal Arg/C-term. Arg128 � bn� 1þH2O Internal Arg/C-term. Lys115 � yn� 1 N-terminal Asp98 � yn–H3PO4 Phospho Ser or Thr80 � yn–HPO3 Phospho Ser or Thr98 � yn–H3PO4 Phospho Tyr80 � yn–HPO3 Phospho Tyr64 � yn–CH3SOH Sulfoxide Met55 � yn–CH2CHCHNH Propionamide Cys44 � yn–CHCH2OH Thr14 � yn–CH2 Methyl Asp or Glu
*The intensity of the mass peak with the mass shift compared to the parent mass is shown.
Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 933–936
934 Letter to the Editor
(Fig. 1(A)). Probably the side chain
(CHCH2OH) of threonine was sepa-
rated from the backbone of the pep-
tides under formation of glycine
(Table 1).
Proteins were observed to be methy-
lated at arginines, lysines and gluta-
mines.12 However, methylation of
aspartic acid or glutamic acid can arise
during the staining procedure of gels in
acidic methanol,13 and was detected
with a mass loss of 14Da compared to
the parent mass (Fig. 2). This mass loss
can only be explained by the reforma-
tion of the carboxylic acid from the
carboxylic ester under loss of CH2
(Table 1). Neutral loss was not
observed for N-terminal acetylated
peptides (Fig. 2).
Metastable decay of phosphopep-
tides by MALDI-MS is well known.14
Because of the tremendous interest in
the detection of phosphorylation sites,
phosphorylated peptides were studied
with synthetic peptides with a C-
terminal lysine, thereby displaying
tryptic peptides, to include the signifi-
cant features into Table 1. Phosphory-
lated serine- and threonine-containing
peptides generated a mass peak at
�98Da with significantly higher inten-
sity compared to the parent mass. In
addition, a mass loss of 80Da was
found also with low intensity
(Figs. 3(A) and 3(B)). The position of
phosphorylated serine and threonine
could be assigned within the fragmen-
tation spectra as dehydrated (�18Da)
amino acidswith themass difference of
69Da for phosphoserine and 83Da for
phosphothreonine. On the other hand,
mass losses of 80 and 98Da with about
the same intensities were observed at
the tyrosine phosphorylated peptide,
both with a lower intensity compared
to the parent mass (Fig. 3(C)). The
phosphorylated amino acid could be
assigned with the mass of 243Da
according to phosphotyrosine.
The prediction of the determined
mass losses was dependent on the
intensity of the mass peaks and the
spectra quality (Table 1). Usually,
equal or higher intensities than the
parent peptide masses were observed
for themass losses of 156, 98, and64Da.
Therefore, internal arginine with C-
terminal arginine, phosphorylation of
serine and threonine, and sulfoxide
of methionine can be unequivocally
determined, or vice versa excluded.
The mass losses due to internal argi-
nine with C-terminal lysine (128Da),
N-terminal aspartic acid (115Da),
phosphorylated tyrosine (98 and
80Da) andmethylation of aspartic acid
or glutamic acid (14Da) displayed
lower intensities than the parent
masses, but were detected in the
majority of the cases. On the other
hand, threonine-containing peptides
(44Da) and propionamide-modified
cysteine-containing peptides (55Da)
were only detected with satisfactory
spectral quality and thus can only be
used as indicators (Fig. 1(C)). As a note,
the individual settings of the MALDI
instruments must be considered. For
example, a MALDI-TOF/TOF mass
spectrometer (4700 Proteomics Analy-
zer, Applied Biosystems) equipped
with a Nd:YAG laser (355 nm) in PSD
mode with metastable suppression of
the parent mass was used here. Never-
theless, the intensities of the described
mass losses should tend to the same
results with different instrument
settings.
37.999
990 1020 1050 1080 1110Mass (m/z)
102030405060708090
100
ytisnet
nI%
55.7901
57.7 101
A
B
C
970 1000 1030 1060 1090Mass (m/z)
45.3801
47.3001
37.589
102030405060708090
100
ytisnet
nI%
1060 1090 1120 1150 1180Mass (m/z)
85.9511
28. 97 01
17.1601
102030405060708090
100
ytisnet
nI%
Figure 3. Distinct mass losses of phosphorylated peptides.
The peptide EAIXAAPFAK with X for phosphoserine (A),
phosphothreonine (B) and phosphotyrosine (C) are depicted.
The neutral loss of 98Da was observed with high intensity for
the peptides with phosphoserine (A) and phosphothreonine (B),
whereas loss of 98 and 80Da with low intensity of the peptide
with phosphotyrosine (C) was detected.
Letter to the Editor 935
Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 933–936
Many MS/MS spectra are not suffi-
cient for unequivocal identification of
known proteins. Furthermore, de novo
sequencing of peptides bymass spectro-
metry requires an unambiguous amino
acid sequence to the fragment spectrum
to obtain high sequence coverage for
database searching. Therefore, any
information within MS/MS spectra
should be used to improve the inter-
pretation. Most of the distinctive mass
losses of tryptic peptides generated by
MALDI-TOF/TOF described in this
report are not integrated into search
algorithms, but can increase the sig-
nificance of sequence-based protein
identifications.
AcknowledgementsWe would like to thank HolgerWenschuh (Jerini, Berlin) for the synth-eses of the phosphorylated peptides.
Frank Schmidt1,2, Alexander Krah2,3,Monika Schmid2, Peter R. Jungblut2 and
Bernd Thiede1*1The Biotechnology Centre of Oslo,
University of Oslo, Gaustadalleen 21,0349 Oslo, Norway
2Max Planck Institute for InfectionBiology, Core Facility Protein Analy-sis, Schumannstrasse 21/22, D-10117
Berlin, Germany3Max Planck Institute for InfectionBiology, Department of MolecularBiology, Schumannstrasse 21/22,
D-10117 Berlin, Germany*Correspondence to: B. Thiede, The Bio-technology Centre of Oslo, University ofOslo, Gaustadalleen 21, P.O. Box 1125Blindern, 0317 Oslo, Norway.E-mail: bernd.thiede@biotek.uio.no
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Received 21 September 2005Revised 9 January 2006
Accepted 9 January 2006
936 Letter to the Editor
Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 933–936