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)

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100

ytisnet

nI%

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100

ytisnet

nI%

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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

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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)

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ytisnet

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55.7901

57.7 101

A

B

C

970 1000 1030 1060 1090Mass (m/z)

45.3801

47.3001

37.589

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nI%

1060 1090 1120 1150 1180Mass (m/z)

85.9511

28. 97 01

17.1601

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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