Báo cáo khoa học: Structural evidence of a-aminoacylated lipoproteins of Staphylococcus aureus pot

Structural evidence of a-aminoacylated lipoproteins of
Staphylococcus aureus
Miwako Asanuma
1,
*, Kenji Kurokawa
2
, Rie Ichikawa
1
, Kyoung-Hwa Ryu
2
, Jun-Ho Chae
2
,
Naoshi Dohmae
1
, Bok Luel Lee
2
and Hiroshi Nakayama
1
1 Biomolecular Characterization Team, RIKEN Advanced Science Institute, Saitama, Japan
2 National Research Laboratory of Defense Proteins, Pusan National University, Busan, Korea
Introduction
Bacterial lipoproteins are lipidated proteins anchored
on the outside leaflet of bacterial cell membranes and
outer envelopes, and have diverse functions such as
nutrient uptake, cell-wall metabolism, adhesion and
transmembrane signaling. The biosynthesis pathway of
bacterial lipoproteins has been established in Escheri-
chia coli and consists of three sequential enzymatic
reactions [1]. Following apolipoprotein translocation
by Sec machinery, the first enzyme diacylglyceryl trans-

ferase (Lgt) transfers a diacylglyceryl moiety from a
membrane phospholipid to a sulfhydryl group of the
+1 cysteine of a conserved ‘lipobox’ motif in the
N-terminal signal peptide, making a thioether linkage.
The lipoprotein signal peptidase (Lsp) then cleaves the
signal peptide at the N-terminus of the +1 S-diacylgly-
ceryl cysteine. Finally, the third enzyme apolipoprotein
Keywords
bacterial lipoprotein; Gram-positive bacteria;
mass spectrometry; N-acyltransferase; SitC
Correspondence
B. L. Lee, National Research Laboratory of
Defense Proteins, College of Pharmacy,
Pusan National University, Jangjeon Dong,
Geumjeong Gu, Busan 609-735, Korea
Fax: +82 51 513 2801
Tel: +82 51 510 2809
E-mail:
H. Nakayama, Biomolecular Characterization
Team, RIKEN Advanced Science Institute, 2-1
Hirosawa, Wako, Saitama 351-0198, Japan
Fax: +81 48 462 4704
Tel: +81 48 462 1419
E-mail:
*Present address
ERATO, Japan Science and Technology
Agency (JST), Tokyo, Japan
(Received 12 November 2010, revised
3 December 2010, accepted 9 December
2010)

doi:10.1111/j.1742-4658.2010.07990.x
Bacterial lipoproteins are known to be diacylated or triacylated and acti-
vate mammalian immune cells via Toll-like receptor 2 ⁄ 6or2⁄ 1 heterodi-
mer. Because the genomes of low G+C content Gram-positive bacteria,
such as Staphylococcus aureus, do not contain Escherichia coli-type apoli-
poprotein N-acyltransferase, an enzyme converting diacylated lipoproteins
into triacylated forms, it has been widely believed that native lipoproteins
of S. aureus are diacylated. However, we recently demonstrated that one
lipoprotein SitC purified from S. aureus RN4220 strain was triacylated.
Almost simultaneously, another group reported that another lipoprotein
SA2202 purified from S. aureus SA113 strain was diacylated. The determi-
nation of exact lipidated structures of S. aureus lipoproteins is thus crucial
for elucidating the molecular basis of host–microorganism interactions.
Toward this purpose, we intensively used MS-based analyses. Here, we
demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipo-
protein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resis-
tant fatty acid. Further MS ⁄ MS analysis of the lipoprotein lipase digest
revealed that the lipoprotein lipase-resistant fatty acid was acylated to
a-amino group of the N-terminal cysteine residue of SitC. Triacylated
forms of SitC with various length fatty acids were also confirmed in cell
lysate of the RN4220 and Triton X-114 phase in three other S. aureus
strains, including SA113 strain and one Staphylococcus epidermidis strain.
Moreover, four other major lipoproteins including SA2202 in S. aureus
strains were identified as N-acylated. These results strongly suggest that
lipoproteins of S. aureus are mainly in the N-acylated triacyl form.
Abbreviations
BHI, Brain Heart Infusion; LB, Luria–Bertani; Lgt, diacylglyceryl transferase; Lnt, apolipoprotein N-acyltransferase; LPL, lipoprotein lipase;
Pam3, N-palmitoyl-S- dipalmitoylglyceryl; TLR, Toll-like receptor; TX114, Triton X-114.
716 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS
N-acyltransferase (Lnt) transfers an acyl group from

phospholipid to the newly generated a-amino group of
the S-diacylglyceryl cysteine (N-acylation reaction),
resulting in the generation of triacylated protein [1,2].
The N-acylation of lipoproteins is essential in Gram-
negative bacteria to transport lipoproteins from the
inner membrane to the outer membrane via the lipopro-
tein localization pathway [3,4].
Although Lgt and LSP are widely conserved in
eubacteria, Lnt has not been found in low G+C
content Gram-positive bacteria (Firmicutes) [5–8].
Recently, Tschumi et al. identified an E. coli Lnt
homolog in the high G+C content Gram-positive bac-
terium Mycobacterium smegmatis and demonstrated its
Lnt activity [9], but its homolog in Firmicutes was not
identified. Several reports have presented structural
and ⁄ or indirect evidence of diacylated lipoproteins, for
example, dipalmitoyl macrophage-activating lipo-
peptide-2 kDa (MALP-2) from Mycoplasma fermen-
tans [10], SA2202 (SAOUHSC_02699) protein from
S. aureus SA113 strain [11] and F
0
F
1
-type ATPase
subunit b from Mycobacterium pneumoniae [12]. There-
fore, Firmicutes are widely regarded as having only
diacylated lipoproteins. Despite the lack of an E. coli
Lnt homolog in Firmicutes, however, chemical analy-
ses of lipoproteins in Bacillus subtilis and in S. aureus
suggested N-acylated lipoproteins in these organisms

[13,14]. We also recently used MS-based analysis to
demonstrate that the SitC lipoprotein from S. aureus is
triacylated [15]; however, we could not show structural
evidence of N-acylation of the lipoprotein. In addition,
some triacylated lipoproteins of Mollicutes, which are
closely related to Firmicutes, have been reported based
upon indirect evidence of nuclear factor-jB activation
through Toll-like receptor (TLR)1 and TLR2 [16,17].
Therefore, evidence of the N-acylation of lipoproteins
leading to triacylated forms in Firmicutes is ambiguous
and controversial.
Microorganism invasion activates the innate immune
response in mammals. Bacterial lipoproteins as a path-
ogen-associated molecular pattern [18] are sensed by
the hosts through TLR2 heterodimerized with TLR1
or TLR6: this signal induces the activation of innate
immunity and is necessary to control adaptive immu-
nity [19]. In addition, TLR2 stimulation drives the dif-
ferentiation of hematopoietic progenitor cells [20].
Although TLR2 has been considered as a receptor for
various structurally unrelated pathogen-associated
molecular patterns such as lipoproteins, lipoteichoic
acid and peptidoglycan [18], recent studies suggest that
bacterial lipoproteins function as the major, if not sole,
ligand molecules for TLR2-activation [5,11,15,21,22].
To date, synthetic lipoprotein analogs, such as N-pal-
mitoyl-S-dipalmitoylglyceryl (Pam3)–Cys, Pam3CSK
4
lipopeptide and MALP-2 [10], have been used to
mimic the proinflammatory properties of bacterial

lipoproteins, and have led to a model in which tria-
cylated lipopeptides signal through TLR2 ⁄ TLR1
heterodimer, whereas diacylated lipopeptides signal
through TLR2 ⁄ TLR6 heterodimer. However, recent
studies have demonstrated that some synthetic lipopep-
tides [23–25] and at least one native lipoprotein [15]
are inconsistent with this model. Therefore, real struc-
tural characterization of native lipoproteins from
Gram-positive bacteria is crucial to elucidate the
molecular basis of host–microorganisms interaction.
Here, we carefully analyzed the structure of the lipo-
peptide moiety of S. aureus lipoproteins. Analyses
using lipoprotein lipase (LPL) and MS ⁄ MS revealed
that S. aureus SitC was N-acylated with various length
fatty acids and thus was triacylated. In addition, SitC
in three other strains of S. aureus and one strain of
S. epidermidis, and four other lipoproteins in S. aureus
were shown to be N-acylated. These results strongly
suggest that lipoproteins in S. aureus are mainly in the
N-acylated triacyl form.
Results
N-Terminal structure of SitC from S. aureus
RN4220 strain
Staphylococcus aureus SitC is annotated as the sub-
strate-binding component of the ATP-binding cassette
transporter for iron [26], and is one of the predomi-
nant lipoproteins functioning as a ligand of TLR2 [27].
Our previous report provided clear MALDI-TOF MS
data representing that the N-terminal lipopeptide of
SitC is triacylated [15]. Although the result is not

enough to support the N-acylation of SitC protein, it
is still surprising because of the presumed absence of
E. coli Lnt homologs in the S. aureus genomes [28].
Contrary to our findings, Tawaratsumida et al.
reported that another lipoprotein SA2202 of S. aureus
SA113 had a diacylated (dipalmitoylated) N-terminus,
based on MS ⁄ MS data [11]. To clarify this discrep-
ancy, we decided to determine the bona fide structure
of lipoproteins in S. aureus. Also, determination of the
exact structure of the Gram-positive bacterial native
lipoproteins is essential for the elucidation of the
molecular mechanism of host–microorganism interac-
tions.
To characterize the acylated structure of S. aureus
lipoproteins, we used commercially available LPL
which is known to degrade bacterial lipoprotein and
reduce the TLR2-stimulating activity of lipoproteins
M. Asanuma et al. Triacylated lipoproteins in S. aureus
FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 717
[21]. At first, we characterized the specificity of the
enzyme using synthetic Pam3CSK
4
triacyl lipopeptide
as a substrate, and found that the enzyme hydrolyzes
O-esterified fatty acids of the (di)acylglyceride moiety,
but not N-acylated fatty acids. To examine the cleav-
age patterns of native SitC lipoproteins by LPL, SitC
protein prepared by Triton X-114 (TX114) phase
partitioning was separated by SDS ⁄ PAGE and then
subjected to in-gel digestion with trypsin to make the

N-terminal lipopeptide of SitC in the presence of
n-decyl-b-D-glucopyranoside, followed by chloro-
form ⁄ methanol extraction. Figure 1A shows MALDI-
TOF MS of the chloroform ⁄ methanol organic phase
representing a series of 14-Da interval peaks, explained
by an increasing number of methylene (CH
2
) groups in
their fatty acids between m ⁄ z 1297 and 1409, which
corresponds with triacylated N-terminal lipopeptides
of SitC (Table 1). The result is consistent with the MS
data from our previous study [15], and suggests that
the N-terminal lipopeptides of SitC were highly puri-
fied, because other peptides generated by the tryptic
digest were rarely detected in the organic phase. As a
positive control, we confirmed that S-dipalmitoylglyce-
ryl–CSK
4
and Pam3CSK
4
lipopeptides were also
recovered in the organic phase using this extraction
method and represented specific signals on MALDI-
TOF MS (data not shown). Recovery of TLR2-stimu-
lating activity in the organic phase was also confirmed
using TLR2-expressing Chinese hamster ovary cells
(Fig. S1), suggesting that the N-terminal lipopeptides
of SitC are responsible for the TLR2 stimulation.
After 5 h incubation with LPL and lipopeptides, a new
series of 14-Da interval peaks was detected between

m ⁄ z 1044 and 1115 (Fig. 1B). The mass values of these
ions corresponds to those of the diacyl-glyceryl
CGTGGK (Table 1) generated by the release of one
O-esterified fatty acid from the original triacylated
lipopeptide. After 17 h incubation, another series of
peaks between m ⁄ z 835 and 891 was detected
(Fig. 1C), corresponding to monoacyl-glyceryl
CGTGGK generated by releasing of two O-esterified
fatty acids from the triacylated lipopeptide (Table 1).
On further incubation, no additional series of peaks
was detected. Thus, the triacylated SitC lipoprotein
1086.75
2
1114.78
1381.02
1
1352.99
1409.05
A
B
1338.98
1072.73
b1
y5
y4
y3
[MH-thioglycerol]
+
[M+H]
+

[MH-2H
2
O]
+
[MH-H
2
O]
+
b°5
b°4
b5
y°5
b°1
0
50
100
Relative intensity
D
[MH-C
2
H
6
O
2
]
+
862.5
754.4
m/z
800 900 100011001200 1300 14001500

862.60
3
890.63
C
300 400 500 600 700 800 900
m/z
NCH
O
C
(C
18
H
35
O)
S
CHHO
CH
2
CH
2
CH
2
HO
H
G T G G K
716.3
698.3
444.5
426.5
362.2

419.3
401.3
b
b°
261.1
y
y°
E
754.4
844.3
826.4
800.5
641.2
Fig. 1. Lipoprotein lipase analysis determines triacylated structures of SitC lipoprotein in Staphylococcus aureus RN4220 cells. SitC lipopro-
tein in the TX114 fraction prepared from exponential-growth phase RN4220 cells was separated by SDS ⁄ PAGE and digested in-gel with tryp-
sin. The resulting N-terminal lipopeptides of SitC were extracted to the organic phase, dried and resolved in water (A) or further incubated
with LPL for 5 h (B) or 17 h (C). MALDI-TOF MS of each fraction is shown. Group 1, 2 or 3 in the figures is consistent with a series of tria-
cylated, diacylated or monoacylated S-glyceryl CGTGGK peptides of N-terminal SitC, respectively, as described in Table 1. The series of
mass signals harboring 14-Da mass differences [an increasing number of methylene (CH
2
) groups] is due to various length saturated fatty
acids. (D) MALDI-IT MS ⁄ MS using 2,5-dihydroxybenzoic acid as a matrix was carried out for an LPL-resistant lipopeptide of m ⁄ z 862.5, as
described in (C). Isolation width was ± 2 Da. (E) The elucidated structure of N-octadecanoly-S-glycerylcysteinyl GTGGK from the observed
fragment ions in (D). Peaks designated y° or b° correspond to y-type or b-type ions that have lost an H
2
O moiety, respectively.
Triacylated lipoproteins in S. aureus M. Asanuma et al.
718 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS
purified from RN4220 cells is modified by two O-ester-
ified fatty acids and one LPL-resistant fatty acid.

To determine the exact modification site of the
LPL-resistant fatty acid, one of the peaks after LPL
digestion corresponding to the octadecanoyl-glyceryl
CGTGGK with m ⁄ z 862.60 shown in Fig. 1C was
further analyzed by MALDI-ion trap (IT) MS ⁄ MS.
Figure 1D,E show the MS ⁄ MS spectrum and the
elucidated structure of the lipopeptide, respectively.
The C-terminus-containing y-series ions at m ⁄ z 261.1
(y
3
), 362.2 (y
4
), 401.3 (y°
5
;y
5
-H
2
O) and 419.3 (y
5
)in
the spectrum confirmed the amino acid sequence of
GTGGK, which is complemented by ions at m ⁄ z
444.5, 641.2 and 698.3, which are assigned as N-
terminus-containing b-series ions (b
1
,b°
4
and b°
5

,
respectively). This result strongly suggests that the
fatty acid modification site is at the N-terminal cyste-
ine residue. Importantly, a characteristic fragment ion
for N-acyl-dehydroalanyl peptide generated by neutral
loss of thioglycerol was observed at m ⁄ z 754.4, indi-
cating that the octadecanoyl group is linked at the
a-amino group of cysteine via an amide bond. These
results are consistent with our previous observation
that the N-terminus of SitC was blocked when
Edman degradation sequencing was performed [15].
Therefore, the results of both LPL digestion and
MS ⁄ MS of the N-terminal peptides demonstrated that
the N-terminal cysteine of SitC from S. aureus
RN4220 cells is N-acylated with a saturated C16 to
C20 fatty acid in addition to the expected S-diacylgly-
cerylation with two saturated fatty acids.
Triacylated SitC is the major form in the cell
lysate of S. aureus RN4220 strain
We next examined whether the triacylated forms are
the major molecular species of SitC in S. aureus cells.
To evaluate the overall molecular species of SitC and
prevent the loss of specific molecular species during
isolation of SitC through the TX114 phase-partitioning
method, the cell lysate of S. aureus RN4220 strain was
directly subjected to SDS ⁄ PAGE (Fig. 2A). When an
approximately 33-kDa band was in-gel digested and
the resulting peptides were analyzed by LC-MS ⁄ MS,
the 33-kDa band was identified as SitC (data not
shown). The digest was then extracted with chloro-

form ⁄ methanol and analyzed by MALDI-TOF MS.
As shown in Fig. 2B, the triacylated N-terminal
lipopeptides of SitC were detected. The arrows indicate
peaks corresponding to triacylated lipopeptides with
the sum of the carbon number for three fatty acids of
47–55 in Table 1. By contrast, any significant peaks
with mass values corresponding to the diacylated
N-terminal lipopeptides of SitC referred to in Table 1
were not detected in the MALDI mass spectrum
(Fig. 2C). These results indicate that the triacylated
forms are the major forms of SitC in S. aureus
RN4220 strain.
Table 1. Calculated and observed masses of the lipid-modified N-terminal peptides of SitC and those generated by lipoprotein lipase diges-
tion shown in Fig. 1.
Modified peptide Calculated [M + H]
+
Observed m ⁄ z D (ppm)
Triacyl(C47) + CGTGGK 1296.94 1296.95 6.4
Triacyl(C48) 1310.96 1310.94 )19.4
Triacyl(C49) 1324.98 1324.95 )21.9
Triacyl(C50) 1338.99 1338.98 )6.5
Triacyl(C51) 1353.01 1352.99 )10.6
Triacyl(C52) 1367.02 1367.01 )12.4
Triacyl(C53) 1381.04 1381.02 )10.6
Triacyl(C54) 1395.05 1395.05 )4.5
Triacyl(C55) 1409.07 1409.05 )12
Diacyl(C30) + CGTGGK 1044.70 1044.71
a
6.3
Diacyl(C31) 1058.72 1058.70

a
)15.2
Diacyl(C32) 1072.73 1072.74
a
12.4
Diacyl(C33) 1086.75 1086.75
a
)0.3
Diacyl(C34) 1100.76 1100.78
a
11.8
Diacyl(C35) 1114.78 1114.78
a
)2.4
Monoacyl(C16) + CGTGGK 834.50 834.57
b
87.4
Monoacyl(C17) 848.52 848.57
b
67.5
Monoacyl(C18) 862.53 862.60
b
78.4
Monoacyl(C19) 876.55 876.61
b
73.0
Monoacyl(C20) 890.56 890.63
b
76.7
Lipoprotein lipase digestion for

a
5 h and
b
17 h.
M. Asanuma et al. Triacylated lipoproteins in S. aureus
FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 719
Characterization of the N-terminal structure of
SitC in other strains of S. aureus and
S. epidermidis
Because one lipoprotein in S. aureus SA113 strain was
reported to be diacylated [11], we then asked whether
the SitC lipoproteins of three other strains of S. aur-
eus, including SA113 strain, and of S. epidermidis
ATCC12228 strain are diacylated or triacylated. The
organic phase of the in-gel-digested SitC isolated from
the TX114 fraction of exponential-growth phase
S. aureus SA113 cells grown in Luria–Bertani (LB)
medium was analyzed by MALDI-TOF MS. As shown
in Fig. 3A and Table 2, a series of 14-Da interval
peaks between m ⁄ z 1283 and 1381, corresponding to
the triacylated N-terminal lipopeptides of SitC modi-
fied with saturated fatty acids (the sum of the carbon
AB
C
Fig. 2. SitC prepared from a crude cell
lysate of S. aureus RN4220 cell is also
triacylated. (A) SDS ⁄ PAGE profile visualized
with Coomassie Brilliant Blue of a crude cell
lysate or its TX114 fraction of S. aureus
RN4220 cells is shown. The arrowhead

indicates the migration position of SitC that
was identified at 33 kDa in either the cell
lysate or TX114 phase. (B,C) MALDI-TOF
mass spectrum of the in-gel tryptic digests
of a 33-kDa region of the cell lysate. Arrows
indicate the calculated mass positions of the
triacylated (B) or diacylated (C) N-terminal
lipopeptides of SitC. Calculated mass values
for the lipopeptides are shown in Table 1.
B
C
A
1367.05
1381.11
1353.05
1339.03
1325.02
1310.99
1296.98
1282.91
*
*
*
*
*
*
C46
C47
C48
C50

C49
C51
C52
C53
1352.99
1381.02
1367.00
1338.98
1324.96
1395.04
1310.95 1409.04
C49
C51
C52
C53
C48
C50
C54
C55
m/z
1367.02
1339.01
1353.01
1324.96
1381.02
1310.97
1280 1300 1320 1340 1360 1380 1400 1420
1409.04
C49
C51

C52
C53
C48
C50
C55
Fig. 3. Triacylated structures of N-terminal peptides of SitC of other
three S. aureus strains. MALDI-TOF mass spectra of the organic
phase of in-gel tryptic digests of SitC protein, which was isolated
through TX114 phase extraction from exponentially growing S. aureus
cells in LB medium of laboratory strain SA113 (A), clinically isolated
methicillin-resistant strain MW2 (B) or clinically isolated methicillin-
sensitive strain MSSA476 (C). Asterisks in (A) are signals 2-Da smaller
than the triacylated peptides filled with saturated fatty acids.
Triacylated lipoproteins in S. aureus M. Asanuma et al.
720 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS
number for three fatty acids is 46–53), was detected. In
the case of clinically isolated S. aureus strains MW2
and MSSA476, a series of 14-Da interval peaks
between m ⁄ z 1311 and 1409, corresponding to the tria-
cyl lipopeptides of SitC with the sum of the carbon
number of the fatty acids equal to 48–55 (see Table 1),
was also detected (Fig. 3B,C). Because Tawaratsumida
et al. used Brain Heart Infusion (BHI) medium when
they determined the N-terminal lipopeptide structure
of SA2202 protein of SA113 strain [11], we used this
medium for the SA113 strain. As in case of LB med-
ium, SitC lipopeptides isolated from SA113 cells grown
in BHI medium had a series of peaks corresponding to
the triacylated forms (data not shown).
The total carbon number of the modified fatty acids

in the most abundant peak of the SitC lipopeptides
derived from the SA113 strain was smaller than that
from RN4220, MW2 or MSSA476 strain, indicating
that shorter fatty acids were mainly attached to the tria-
cylated lipopeptides of SitC of the SA113 strain
(Figs 1A and 3). The usage of shorter fatty acids was
also detected in the spectrum of the SitC lipopeptides
isolated from SA113 cells grown in BHI medium (data
not shown). Moreover, triacyl peptides 2 Da smaller
than those loaded with saturated fatty acids were addi-
tionally observed in SA113 cells grown in both LB and
BHI medium, and are indicated by asterisks in Fig. 3A.
These peaks would be due to the presence of an unsatu-
rated fatty acid in the triacylated lipopeptides.
Table 2. Calculated and observed masses of triacylated N-terminal
lipopeptides of SitC and SA2202 isolated from exponentially grow-
ing S. aureus SA113 cells grown in Luria-Bertani medium.
Modified peptide Calculated [M + H]
+
Observed m ⁄ z D (ppm)
C46 + CGTGGK
a
1282.93 1282.91 )15.6
C47 1296.94 1296.98 30.8
C48 1310.96 1310.99 22.9
C49 1324.98 1325.02 30.2
C50 1338.99 1339.03 29.9
C51 1353.01 1353.05 29.6
C52 1367.02 1367.05 21.9
C53 1381.04 1381.11 50.7

C45 + CGNNSSK
b
1455.97 1456.01 23.1
C46 1469.99 1470.04 30.6
C47 1484.01 1484.06 33.3
C48 1498.02 1498.07 31.2
C49 1512.04 1512.09 37.1
C50 1526.05 1526.12 43.5
C51 1540.07 1540.12 34.9
C52 1554.08 1554.16 48.3
C53 1568.10 1568.12 15.6
C54 1582.12 1582.16 27.7
a
SitC,
b
SA2202.
200 400 600 800 1000 1200
A
B
H
+
C
O
H
+
O
CR
3
CH
2

S
CH
2
CHO
CH
2
O
R
2
R
1
H
peptide
NCH
NCH
O
CR
3
CH
2
S
CH
2
CHO
R
2
OCH
2
R
1

H
peptide
+ H
2
O
2
Energy
C17
C17
C15
1339.0
*
[M+H]
+
[M+H]
+
y2
y3
y4
y5
y1
y°1
Relative intensity
*
m/z
B
NC
O
CR
3

CH
2
H
peptide
H
+
N-acyl-dehydroalanyl peptide ion
S
CH
2
CH
O
R
2
OCH
2
R
1
HO
2,3-diacyloxypropane
sulfenic acid
Neutral loss
200 400 600 800 1000 1200
1355.0
y2
y3
y4
y5
C15
C18

C16
C19
C20
dAGTGGK-NH
3
dAGTGGK
Relative intensity
Fig. 4. Staphylococcus aureus SA113 cells also have the N-acylated triacyl-forms of SitC lipoprotein. (A) SitC lipoprotein of exponentially
growing S. aureus SA113 cells in BHI medium was prepared as in Fig. 1A. Among the observed N-terminal lipopeptides of SitC similar to
those described in Table 2 (LB medium) a signal at m ⁄ z 1339.0 corresponding to C50-triacylated lipopeptides was analyzed by MALDI-TOF
MS ⁄ MS. Asterisks indicate molecular ion peaks generated by the neutral loss of diacylthioglycerol. (B) Lipopeptides used in (A) were oxi-
dized by spotting hydrogen peroxide solution on to the sample–matrix co-crystal before MALDI-TOF MS. MALDI-TOF MS ⁄ MS spectrum of
the on-target oxidized C50-triacylated lipopeptide at m ⁄ z 1355.0 is shown. Signals marked with C15 to C20 indicate the N-acylated dehydro-
alanyl peptide ions generated by neutral loss of 2,3-diacyloxypropane-1-sulfenic acid having different length fatty acids, as indicated by
carbon numbers. Peaks designated dAGTGGK or dAGTGGK-NH
3
correspond to the dehydroalanyl peptide ion or its de-ammonium ion,
respectively, which are secondarily generated from N-acyl-dehydroalanyl peptide ions by losing fatty acid. (C) Reaction scheme of H
2
O
2
oxidation and collision-induced dissociation. Incorporated oxygen, shown in bold, adds 16 Da to lipopeptides, such as from m ⁄ z 1339.0 (A)
to m ⁄ z 1355.0 (B). The rectangle indicates dehydroalanine. R1, R2 and R3 indicate a hydrogen or acyl group.
M. Asanuma et al. Triacylated lipoproteins in S. aureus
FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 721
To prove the N-acylated structure of SitC from
other than the RN4220 strain, N-terminal lipopeptide
from the SA113 strain grown in BHI medium was ana-
lyzed by MS ⁄ MS. Figure 4A shows the MS ⁄ MS
spectrum of the peak at m ⁄ z 1339.0, corresponding to

triacylated lipopeptide in which the sum of the carbon
number for three fatty acids is 50. The y-series ions
detected in the spectrum are essentially the same as
those from the SitC lipopeptide from RN4220
(Fig. 1D), confirming that the peptide moiety of N-ter-
minus is identical between the two strains. Weak peaks
around m ⁄ z 750 corresponding to N-acyl-dehydroala-
nyl peptide ions generated by the neutral losses of dia-
cylthioglycerol moieties, the hallmark of N-acylation,
were also detected in the spectrum (Fig. 4A). Because
these characteristic peaks are usually weak, and some-
times not detectable, we developed a method that can
sensitively detect the neutral losses. Figure 4B shows
MS ⁄ MS spectrum of the lipopeptide of SitC on-target
oxidized with H
2
O
2
, in which intense peaks between
m ⁄ z 712 and 782 were obtained. The increase in
N-acylated dehydroalanyl peptide ions is explained by
the fact that oxidized lipoproteins undergo facile neu-
tral loss of 2,3-diacyloxypropane-1-sulfenic acid in
MS ⁄ MS (or MALDI-MS) (Fig. 4C). The reaction
mechanism should be similar to the neutral loss of
methane sulfenic acid from methionine sulfoxide by
MS ⁄ MS [29]. The result shown in Fig. 4B clearly dem-
onstrates that a saturated fatty acid (from C15 to C20)
is linked at the a-amino group of SitC.
In addition to these S. aureus strains, analysis of

SitC purified from S. epidermidis [26] gives a series of
14-Da interval peaks between m ⁄ z 2512 and 2597, cor-
responding to the N-terminal lipopeptides of the triacy-
lated SitC (Fig. 5A). Of these, the peak at m ⁄ z 2568.5,
corresponding to triacylated CGNHSNHEHHSHEGK
(the sum of the carbon number for three fatty acids is
53), was analyzed by MALDI-TOF MS ⁄ MS. The
MS ⁄ MS spectrum shows the characteristic N-acyl
dehyroalanyl peptide ions with C17 to C20 saturated
fatty acid (Fig. 5B), and its elucidated structure
strongly indicates that the S. epidermidis SitC lipopro-
tein is also in the N-acylated triacyl form (Fig. 5C).
Characterization of other lipoproteins of
S. aureus
Because SA2202 lipoprotein in S. aureus SA113 strain
was reported to be diacylated with two palmitic acids
rather than with different length fatty acids [11], we
examined whether SA2202 in the SA113 strain was
triacylated and also examined the composition of the
fatty acids. Using the methods described above for
2568.49
2596.522554.45
2582.51
2540.44
2526.41
2512.41
2520 2540 2560 2580 2600 2620
Relative intensity
C53
C52

C51
C50
C49
C54
C55
A
m/z
m/z
y5
y6
y7
y8
y9
y10
y11
y12
y14
C18
C17
C19
C20
[M+H]
+
2568.4
500 1000 1500 2000 2500
Relative intensity
B
C
R
1

R
2
GN H
N
HE
H H SHEGK
y
O
N CH
CH
2
CH
S
CH
2
CH
2
O
O
n = 20: 1968.2
19: 1954.5
18: 1940.3
17: 1926.3
R
3
H
C
R
1
: C

l
H
2l-1
O
R
2
: C
m
H
2m-1
O
R
3
: C
n
H
2n-1
O
1606.5
1435.5
1299.7
1212.2
1097.9
959.9
831.2
694.3
557.0
S
Fig. 5. N-Acylated triacyl structure of S. epidermidis SitC. (A)
MALDI-TOF mass spectrum of an organic phase of in-gel tryptic

digest of SitC, isolated through TX114 phase extraction from expo-
nential-growth phase S. epidermidis ATCC12228 cells in LB med-
ium. The sum of carbon numbers for three fatty acids is labeled at
the peaks corresponding to the monoisotopic mass values of the
triacylated N-terminal lipopeptides of SitC (CGNHSNHEHHSHEGK).
(B) MALDI-TOF MS ⁄ MS spectrum of the precursor ion of
m ⁄ z 2568.5 (C53) shown in (A). Signals marked C17 to C20 indi-
cate the N-acylated dehydroalanyl peptide ions generated by neutral
loss and with different length saturated fatty acids with the
indicated carbon numbers. (C) The elucidated structure of C53-tria-
cylated N-terminal lipopeptides of S. epidermidis SitC with assign-
ments of the observed fragment ions in panel B. The l, m and n
are positive integers and l + m + n = 53.
Triacylated lipoproteins in S. aureus M. Asanuma et al.
722 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS
SitC in RN4220 strain, lipopeptides of SA2202 from
the SA113 strain were prepared and analyzed by
MALDI-TOF MS. As shown in Table 2, characteristic
peaks corresponding to triacyl N-terminal lipopeptides
of SA2202 with a total of 45 to 54 carbon numbers of
three saturated fatty acids were detected in the
MALDI-TOF mass spectrum of the in-gel tryptic
digest itself and its organic phase extract, but diacyl
lipopeptide signals were not. Like SitC, SA2202 pro-
tein in the SA113 strain was modified, mainly with
shorter fatty acids than in RN4220 (Tables 2 and 3)
and peaks containing unsaturated fatty acids were also
observed (data not shown). Figure 6A,B shows the
MS ⁄ MS spectrum and the elucidated structure of the
most abundant peak, corresponding to the oxidized

C50-triacyl lipopeptides. The spectrum clearly showed
the characteristic N-acyl-dehydroalanyl peptide ions
with C15 to C20 saturated fatty acid, suggesting that
the SA2202 lipoprotein in SA113 is the N-acylated
triacyl form with different length fatty acids.
We then asked whether other lipoproteins in the
RN4220 strain were N-acylated. To address this, we
searched for other lipoproteins in the TX114 phase of
S. aureus RN4220. LC-MS ⁄ MS of the in-gel tryptic
Table 3. Calculated and observed masses of the triacylated N-
terminal lipopeptides of SA2202, SA0739, SA0771, SA2074, and
SA2158 proteins isolated from exponentially growing S. aureus
RN4220 cells grown in LB medium.
Modified peptide Calculated [M+H]
+
Observed m ⁄ z D (ppm)
C48 + CGNNSSK
a
1498.02 1497.94 )53.2
C49 1512.04 1511.94 )63.0
C50 1526.05 1525.97 )53.0
C51 1540.07 1539.98 )56.2
C52 1554.08 1554.00 )52.8
C53 1568.10 1568.01 )55.9
C54 1582.11 1582.03 )52.6
C55 1596.13 1596.05 )49.4
C44 + CGHHQDSAK
b
1715.08 1715.01 )42.8
C45 1729.10 1729.04 )33.9

C46 1743.11 1743.11 )1.4
C47 1757.13 1757.16 17.2
C48 1771.14 1771.14 1.3
C49 1785.16 1785.15 )5.6
C50 1799.17 1799.17 )1.2
C51 1813.19 1813.17 )11.7
C52 1827.20 1827.17 )19.1
C47 + CGNGNK
c
1366.96 1366.95 )8.4
C48 1380.98 1380.98 2.1
C49 1394.99 1394.95 )30.6
C50 1409.01 1408.98 )20.1
C51 1423.02 1422.95 )51.9
C52 1437.04 1436.96 )55.3
C53 1451.06 1450.98 )51.7
C54 1465.07 1464.97 )68.7
C55 1479.09 1478.97 )78.6
C49 + CSNSNDNNESK
d
2014.20 2014.15 )25.5
C50 2028.22 2028.16 )28.0
C51 2042.23 2042.22 )6.1
C52 2056.25 2056.24 )3.9
C53 2070.26 2070.24 )11.4
C54 2084.28 2084.27 )4.5
C55 2098.29 2098.26 )16.6
C45 + CGQDSDQQK
e
1755.08 1755.11 16.6

C46 1769.10 1769.14 20.8
C47 1783.12 1783.13 8.5
C48 1797.13 1797.16 13.3
C49 1811.15 1811.15 0.5
C50 1825.16 1825.19 12.6
C51 1839.18 1839.17 )3.9
C52 1853.19 1853.18 )5.1
a
SA2202,
b
SA0739,
c
SA0771,
d
SA2074,
e
SA2158.
R
1
S
CH
2
CHO
OCH
2
R
2
C17
1542.0
[M+H]

+
y2
y3
y4
y5
C15
C18
C16
C19
C20
y6
200 600 1000 1400
m/z
Relative intensity
R
1
: C
l
H
2l-1
O
R
2
: C
m
H
2m-1
O
R
3

: C
n
H
2n-1
O
O
N
CH
O
C
CH
2
S
H
R
3
G N N S S K
234.0321.1435.1
y
549.1606.1
n = 20: 969.5
19: 955.5
18: 941.5
17: 927.5
16: 913.5
15: 899.5
O
A
B
Fig. 6. Lipoprotein SA2202 of S. aureus SA113 shows N-acylated

triacyl-forms. (A) MALDI-TOF MS ⁄ MS was carried out for N-termi-
nal lipopeptides of SA2202 lipoprotein from exponentially growing
SA113 cells as described in Fig. 4B. Precursor peaks used were
molecular ions of the on-target oxidized C50-triacylated lipopeptide
at m ⁄ z 1542.0. Signals marked with C15 to C20 indicate the N-acyl-
ated dehydroalanyl peptide ions that were generated by neutral loss
and had different length fatty acids with the indicated carbon num-
bers. (B) Elucidated structures of the oxidized N-terminal triacyl lipo-
peptides. R1, R2 and R3 indicate acyl group. The l, m and n are
positive integers and l + m + n = 50. The value of n was deter-
mined to be from 15 to 20 (A). Calculated mass values of the N-ter-
minal triacyl lipopeptides of SA2202 are found in Table 2.
M. Asanuma et al. Triacylated lipoproteins in S. aureus
FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 723
digest of a minor band enabled us to identify five other
lipoproteins, SA0739, SA0771, SA2074 (ModA),
SA2158 and SA2202. To concentrate these lipoproteins,
the Zn-stained lipoproteins on the acrylamide gels were
collected and then eluted from gel pieces by simple dif-
fusion, concentrated and resubjected to SDS ⁄ PAGE.
The concentrated protein band was then in-gel digested.
Using the methods described above for SitC, the result-
ing lipopeptides were extracted by chloroform ⁄ metha-
nol and analyzed by MALDI-TOF MS. Table 3 shows
the observed and calculated masses of these lipopeptide
peaks in each lipoprotein. A series of these peaks at an
interval of m ⁄ z 14 in each protein corresponded to
the calculated molecular mass values of triacylated
N-terminal lipopeptides of each. In contrast, mass
signals corresponding to diacylated lipopeptides were

not observed (data not shown). Therefore, these five
lipoproteins are suggested to be mainly triacylated.
N-Acylation of these triacylated lipoproteins from
RN4220 was further demonstrated by MALDI-TOF
MS ⁄ MS (Fig. 7). Regarding SA2202 protein, Fig. 7A
shows the MS ⁄ MS spectrum of the most abundant peak
at m ⁄ z 1553.9, corresponding to the N-terminal tria-
cylated CGNNSSK lipopeptide (the sum of the carbon
number for three fatty acids was 52; see Table 3). The
oxidized lipopeptides were also analyzed by MS ⁄ MS
(shown as an inset in Fig. 7A). Both spectra represented
the characteristic N-acyl-dehydroalanyl peptide ions
due to neutral loss, whose signals were enhanced in the
oxidized lipopeptides. The fatty acid linked to the
a-amino group was a saturated fatty acid with a length
of C16 to C20. Likewise, two lipoproteins (SA0739 and
SA0771) were also successfully determined to be N-acyl-
ated due to the detection of the N-acyl-dehydroalanyl
peptides caused by the neutral losses using the oxidized
lipopeptides (Fig. 7B,C). Figure 7D shows the MS ⁄ MS
spectrum of the triacylated N-terminal lipopeptide of
SA2074, which presents relatively week but significant
peaks of the N-acyl-dehydroalanyl peptide ions (C15–
C20) generated by the neutral loss of diacylthioglycerol,
indicating N-acylation of the lipopeptide. An MS ⁄ MS
spectrum of the triacylated lipopeptides of SA2158 pro-
tein did not show significant signals because of the low
intensity of the lipopeptide peaks.
Discussion
This study presents, for the first time, the structure of

N-terminal lipids of native S. aureus lipoproteins. Here,
we provide solid structural evidence for N-acylated
triacyl forms of SitC and four other lipoproteins in
S. aureus RN4220 using intensive MS-based analysis,
combined with LPL or H
2
O
2
treatment. The triacylated
lipoproteins were confirmed in other three S. aureus
strains including SA113 and in one S. epidermidis
strain, strongly suggesting that lipoproteins of S. aureus
are mainly N-acylated triacyl forms. Lipoproteins in
Firmicutes were thought to be diacylated because of
the absence of E. coli-type Lnt in these bacteria [22];
the hypothesis was that immune cells could discrimi-
nate between Gram-negative and Gram-positive bacte-
ria by the ability of TLR2 to form heterodimers with
200 400 600 800 1000 1200 1400
m/z
C
B
A
HQ
HQD
y5
y4
y6
y7
y8

b5
b6
[M+H]
+
1827.12
200 400 600 800 1000 1200 1400 1600 1800
Relative intensity
C17
C18
1160 1200 1240
C15
C16
C19
C20
[M+H]
+
y3
y4
y6
y5
y2
y1
1553.9
C17
C18
900 940 980
C16
C19
C20
200 400 600 800 1000 1200 1400

Relative intensity
C
[M+H]
+
y2
y4
y5
y6
y7
y8
y10
y9
2043.37
200 400 600 800 1000 1200 1400 1600 1800 2000
Relative intensity
[M+H]
+
y5
y4
y3
y2
y°1
1437.10
200 400 600 800 1000 1200 1400
Relative intensity
m/z
800 840 880
C16
C17
C18

C19
C20
14401400 1480
C17
C16
C18
C19
C15
C20
D
Fig. 7. Other N-acylated triacyl-lipoproteins of S. aureus RN4220
cells. MALDI-TOF MS ⁄ MS spectrum of N-terminal lipopeptides of
SA2202 protein with C52 (A), SA0739 protein with C52 (B),
SA0771 protein with C52 (C) or SA2074 protein with C51 (D)
prepared from exponential-growth phase S. aureus RN4220 cells in
LB as SitC of Fig. 1A. The mass of the precursor ion for each is
described in Table 3. The inset in each panel is the MALDI-TOF
MS ⁄ MS spectrum of on-target oxidized lipopeptides for each (A–C)
or the magnified view (D). N-Acyl-dehydroalanyl peptide ions gener-
ated by the neutral loss of 2,3-diacyloxypropane-1-sulfenic acid or
diacylthioglycerol were observed and are indicated by C15 to C20
in the insets. Peaks designated with HQ and HQD in (B) are
internal fragment ions.
Triacylated lipoproteins in S. aureus M. Asanuma et al.
724 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS
TLR1 or TLR6, in response to triacyl lipopeptides or
diacyl lipopeptides, respectively [30]. However, our
study provides some clear evidence that these predic-
tions may need to be reconsidered.
Contrary to our results, Hashimoto’s group reported

that the N-terminus of SA2202 lipoprotein in S. aureus
SA113 was only an S-dipalmitoylglyceryl cysteine form
in S. aureus SA113 [11]. Discrepancies with our results
are likely due to differences in cell-culture, lipoprotein
preparation or analysis methods, because the SA113
strain we used was received from Hashimoto’s group.
Differences in method resulted in several hundred-fold
differences in the recovery of lipoprotein for structural
analysis; their previous overall yield was only 1.6 lgof
each lipoprotein per l-culture [11], whereas we obtained
several hundred micrograms of SitC in our TX114
phase per l-culture (data not shown). In addition, we
developed the analytical method including in-gel diges-
tion and organic solvent extraction in the presence of
0.1% n-decyl-b-D-glucopyranoside (see Experimental
procedures), which prevents the loss of very hydropho-
bic lipopeptides. As a consequence, we detected a series
of peaks modified with different length saturated and
unsaturated fatty acids. Because phospholipids in bac-
teria have various length fatty acids and Lgt transfers
a diacylglycerol moiety from phospholipids to the lipo-
protein precursor, it is reasonable to conclude that
lipoproteins are modified with different length fatty
acids. Therefore, detection of only the dipalmitoyl-
glyceryl form in SA2202 [11] indicates insufficient sensi-
tivity to detect heterogeneity of the acylation in their
analysis. Further, we showed the predominance of tria-
cylated SitC in cell lysate (Fig. 2). Therefore, we believe
that we have obtained unbiased results, and that the
major form of lipoproteins in S. aureus is the triacylat-

ed one. Recently, a similar procedure with us allowed
the detection of triacylated lipopeptides of LppX lipo-
protein from M. smegmatis [9]. However, our results
do not rule out the existence of diacylated lipoproteins
in the bacterium. Because they are intermediate forms
during biosynthesis of the triacylated lipoproteins, they
are likely to be a minor component under our condi-
tions. In fact, in addition to mass signals of the tria-
cylated form, relatively weak signals corresponding to
diacylated lipopeptides of SitC with various fatty acids
were also detected from high-temperature culture (data
not shown), suggesting that the degree of N-acylation
may depend on bacterial growth conditions.
Our results also suggest that unsaturated fatty acid
was incorporated in lipoproteins of S. aureus SA113
strain. Although further analysis to determine the
modification site(s) and molecular species of the unsat-
urated fatty acid in bacterial lipoproteins is required,
its roles in ligand recognition and receptor activation
for TLR2 are curious.
In addition to our studies, several reports provide
indirect evidence of triacylated lipoprotein(s) in Firmi-
cutes [13,14] and Mollicutes [16,17]. Although Firmi-
cutes do not have an E. coli Lnt homolog [5–8], our
results strongly suggest that S. aureus and also S. epide-
rmidis have an unidentified enzyme which can catalyze
the N-acylation of diacylated lipoproteins with a satu-
rated fatty acid, whose structure is distinct from E. coli
and M. smegmatis Lnt. N-Acylation of lipoproteins in
E. coli is characterized as being required for lipoprotein

localization machinery LolCDE-dependent release of
outer membrane-specific lipoproteins from the inner
membrane [3], and deficiency of Lnt is known to cause
mislocation of lipoproteins [31]. Identification of the
unidentified N-acylation enzyme in S. aureus should
facilitate understanding of the biological significance of
the lipoprotein N-acylation.
Experimental procedures
Bacterial strains, plasmids, and culture
conditions
The S. aureus SA113 strain was a gift from Dr M. Hashim-
oto (Kagoshima University, Japan). The methicillin-resistant
S. aureus MW2 strain and methicillin-sensitive S. aureus
MSSA476 strain were obtained from the Network on Anti-
microbial Resistance in Staphylococcus aureus (Chantilly,
VA). The Staphylococcus epidermidis ATCC12228 strain was
obtained from the Korean Culture Center of Microorgan-
isms (Seoul, Korea). These S. aureus and S. epidermidis
strains were grown in LB or BHI medium at 37 °C until late
exponential-growth phase, as described previously [15].
Purification of lipoproteins by TX114 phase
partitioning
Lipoproteins were obtained using the TX114 phase-parti-
tioning method, as described previously [15]. Briefly, the
harvested S. aureus cells were disrupted with glass beads
and centrifuged at 2000 g for 10 min to remove the glass
beads and unbroken cells, and the resulting supernatant
was further centrifuged at 20 000 g for 10 min to remove
the peptidoglycan fraction. The obtained supernatant was
used as a cell lysate, or was supplemented with TX114 to a

final concentration of 2%, and then incubated at 4 °C for
1 h. The mixture was subsequently incubated at 37 °C for
10 min for phase separation. After centrifugation at
10 000 g for 10 min at 25 °C, the upper aqueous phase was
removed and was replaced with the same volume of a
TBSE solution (20 mm Tris ⁄ HCl, pH 8, 130 m m NaCl and
5mm EDTA). This procedure was repeated twice. The final
M. Asanuma et al. Triacylated lipoproteins in S. aureus
FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS 725
TX114 phase was precipitated with ethanol and precipitates
were used for subsequent experiments as a TX114 phase.
In-gel digestion
Proteins in the TX114 phase were separated by SDS ⁄ PAGE
and stained with Coomassie Brilliant Blue R250. The pro-
tein bands excised from the gel were destained with
50% (v ⁄ v) methanol and dried by vacuum centrifugation.
The dried gel pieces were rehydrated with 2 lLof
10 ngÆlL
)1
trypsin (Promega, Madison, WI) and then incu-
bated in  20 lLof50mm Tris ⁄ HCl (pH 8.5) containing
0.1% (w ⁄ v) n-decyl-b-d-glucopyranoside (Sigma, St. Louis,
Mo) at 37 ° C for 18 h [32]. The resulting digests were ana-
lyzed by MS directly or after chloroform ⁄ methanol extrac-
tion. Note that the sample tubes used for in-gel digestion
and subsequent analytical procedures were hydrophilic
polypropylene tubes (Proteosave SS, Sumitomo Bakelite,
Tokyo, Japan) to decrease the loss of lipoproteins or lipo-
peptides by nonspecific adsorption to the tube.
Purification of lipopeptides by chloroform

⁄
methanol extraction
Lipopeptides from the purified native lipoproteins were
extracted according to the method of Ujihara et al. [33]
with some modifications. In brief, a peptide solution
(10–20 lL) resulting from in-gel digestion was acidified with
1 lL of 70% formic acid and then mixed with the same
volume of a chloroform ⁄ methanol (2 : 1, v ⁄ v) solvent by
vortexing and centrifuged at 10 000 g for 10 min to sepa-
rate into an organic (lower) and an aqueous (upper) phase.
The organic phase containing lipopeptides was transferred
to a new tube. It is noteworthy that the acidification step is
necessary because, in the presence of tryptic activity, forma-
tion of methylester at the C-terminal carboxyl group of
lipopeptides is often observed in the organic phase.
MALDI MS and MS
⁄
MS
MALDI-TOF MS was conducted using an Ultraflex (Bru-
ker Daltonics, Bremen, Germany) in positive reflectron
mode. Saturated a-cyano-4-hydroxycinnamic acid (CHCA)
solution in a chloroform ⁄ methanol (2 : 1, v ⁄ v) solvent was
used as matrix. A thin layer of CHCA matrix was prepared
and the samples were deposited on the matrix. MS ⁄ MS
spectra were acquired using a MALDI-IT mass spectro-
meter (MALDI-LTQ, Thermo Fisher Scientific, Waltham,
MA) or MALDI-TOF ⁄ TOF instrument (Ultraflex; Bruker
Daltonics) using CHCA or 2, 5-dihydroxybenzoic acid as
matrix. Fragments of tryptic lipopeptides observed in
MALDI MS ⁄ MS spectrum are mainly C-terminus-contain-

ing sequence ions (y-type) and less intense N-terminus-
containing ions (b-type) because positive charge tends to
localize at C-terminal lysine or arginine of the peptides.
On-target oxidation of lipopeptides
To facilitate neutral loss of diacylglyceride moiety from
lipopeptides by MS ⁄ MS, thioether sulfur at the N-terminal
diacylglycerylcysteine of a lipopeptide was oxidized to sulf-
oxide with hydrogen peroxide. One microliter of hydrogen
peroxide (30% aqueous solution) was spotted onto the sam-
ple–matrix co-crystal on a MALDI sample target and the
target was stood at room temperature until dry. Oxidation
could also be done before deposition to the sample target.
The hydrogen peroxide solution was added to a sample
solution (final concentration 10%) and the mixture was
incubated at 37 °C for 1 h.
LPL treatment for lipopeptides
Organic phase containing lipopeptides was dried under
vacuum and the lipopeptides were resolved in 10–20 lLof
water by sonication for 5 min. The solution was heated at
100 °C for 1.5 h to inactivate trypsin and incubated at
37 °C with 80 ngÆlL
)1
LPL from Pseudomonas sp. (Sigma).
The resulting digests were directly analyzed by MALDI MS
and MS ⁄ MS.
Protein identification by liquid chromatography
(LC)-MS
⁄
MS
In-gel digests were analyzed by a nano-LC (1100 series;

Agilent Technologies, Santa Clara, CA) coupled with a
hybrid quadrupole-TOF instrument (Q-Tof2; Waters, Mil-
ford, MA) as described previously [32]. The mobile phases
of a home-made capillary column packed with Inertsil
ODS3 (GL Science, Tokyo, Japan) consisted of solvent A,
0.075% (v ⁄ v) formic acid in water, and solvent B, 0.075%
(v ⁄ v) formic acid and 80% (v ⁄ v) acetonitrile in water. The
resulting MS ⁄ MS data were applied to Mascot (Matrix
Science, London, UK) for the protein identification. The
gene IDs for protein identification used here were from
S. aureus strain N315 strain [7].
Acknowledgements
We thank Dr Masahito Hashimoto of Kagoshima
University for providing S. aureus SA113 strain and
Dr Youngnim Choi of Seoul National University for
help with analysis of TLR2-expressing CHO. We also
thank Mr Daisuke Higo of Thermo-Fischer Scientific
and Dr Ryo Taguchi of University of Tokyo for help
with MALDI-IT MS ⁄ MS measurement.
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Supporting information
The following supplementary material is available:
Fig. S1. TLR2 stimulation by triacylated N-terminal
SitC lipopeptide from S. aureus RN4220 cells.
This supplementary material can be found in the
online version of this article.
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Triacylated lipoproteins in S. aureus M. Asanuma et al.
728 FEBS Journal 278 (2011) 716–728 ª 2011 The Authors Journal compilation ª 2011 FEBS