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-

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

[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

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

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]
+

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

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

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
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

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

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

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

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

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

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

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

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 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]

: 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

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

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

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

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

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

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;

3 Fukuda A, Matsuyama S, Hara T, Nakayama J,
Nagasawa H & Tokuda H (2002) Aminoacylation of
the N-terminal cysteine is essential for Lol-dependent
release of lipoproteins from membranes but does not
depend on lipoprotein sorting signals. J Biol Chem 277,
43512–43518.
4 Tokuda H & Matsuyama S (2004) Sorting of lipopro-
teins to the outer membrane in E. coli. Biochim Biophys
Acta 1693, 5–13.
5 Stoll H, Dengjel J, Nerz C & Go
¨
tz F (2005) Staphylo-
coccus aureus deficient in lipidation of prelipoproteins is
attenuated in growth and immune activation. Infect
Immun 73, 2411–2423.
6 Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni
G, Azevedo V, Bertero MG, Bessie
`
res P, Bolotin A,
Borchert S et al. (1997) The complete genome sequence
of the Gram-positive bacterium Bacillus subtilis. Nature
390, 249–256.
7 Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Ogu-
chi A, Nagai Y, Iwa m a N , Asano K, Naimi T et al. (2002)
Genome and virulence determinants of high virulence
community-acquired MRSA. Lancet 359, 1819–
1827.
8 Paulsen IT, Banerjei L, Myers GSA, Nelson KE, Sesh-
adri R, Read TD, Fouts DE, Eisen JA, Gill SR, Heidel-
berg JF et al. (2003) Role of mobile DNA in the

14 Navarre WW, Daefler S & Schneewind O (1996) Cell
wall sorting of lipoproteins in Staphylococcus aureus.
J Bacteriol 178, 441–446.
15 Kurokawa K, Lee H, Roh KB, Asanuma M, Kim YS,
Nakayama H, Shiratsuchi A, Choi Y, Takeuchi O,
Kang HJ et al. (2009) The triacylated ATP binding
cluster transporter substrate-binding lipoprotein of
Staphylococcus aureus functions as a native ligand for
Toll-like receptor 2. J Biol Chem 284, 8406–8411.
16 Shimizu T, Kida Y & Kuwano K (2007) Triacylated
lipoproteins derived from Mycoplasma pneumoniae
activate nuclear factor-jB through toll-like receptors 1
and 2. Immunology 121 , 473–483.
17 Shimizu T, Kida Y & Kuwano K (2008) A triacylated
lipoprotein from Mycoplasma genitalium activates
NF-jB through Toll-like receptor 1 (TLR1) and TLR2.
Infect Immun 76, 3672–3678.
18 Takeuchi O & Akira S (2010) Pattern recognition recep-
tors and inflammation. Cell 140, 805–820.
19 Iwasaki A & Medzhitov R (2004) Toll-like receptor
control of the adaptive immune responses. Nat Immunol
5, 987–995.
20 Nagai Y, Garrett KP, Ohta S, Bahrun U, Kouro T,
Akira S, Takatsu K & Kincade PW (2006) Toll-like
receptors on hematopoietic progenitor cells stimulate
innate immune system replenishment. Immunity 24,
801–812.
21 Hashimoto M, Tawaratsumida K, Kariya H, Aoyama
K, Tamura T & Suda Y (2006) Lipoprotein is a predomi-
nant Toll-like receptor 2 ligand in Staphylococcus aureus

27 Mu
¨
ller P, Mu
¨
ller-Anstett M, Wagener J, Gao Q, Kaes-
ler S, Schaller M, Biedermann T & Go
¨
tz F (2010) The
Staphylococcus aureus lipoprotein SitC colocalizes with
Toll-like receptor 2 (TLR2) in murine keratinocytes and
elicits intracellular TLR2 accumulation. Infect Immun
78, 4243–4250.
28 Hutchings MI, Palmer T, Harrington DJ & Sutcliffe IC
(2009) Lipoprotein biogenesis in Gram-positive bacteria:
knowing when to hold ‘em, knowing when to fold ‘em.
Trends Microbiol 17, 13–21.
29 Lagerwerf FM, van de Weert M, Heerma W & Hav-
erkamp J (1996) Identification of oxidized methionine
in peptides. Rapid Commun Mass Spectrom 10 , 1905–
1910.
30 Schenk M, Belisle JT & Modlin RL (2009) TLR2 looks
at lipoproteins. Immunity 31 , 847–849.
31 Robichon C, Vidal-Ingigliardi D & Pugsley AP (2005)
Depletion of apolipoprotein N-acyltransferase causes
mislocalization of outer membrane lipoproteins in
Escherichia coli. J Biol Chem 280, 974–983.
32 Kodama K, Fukuzawa S, Nakayama H, Kigawa T,
Sakamoto K, Yabuki T, Matsuda N, Shirouzu M,
Takio K, Tachibana K et al. (2006) Regioselective
carbon–carbon bond formation in proteins with palla-