Tài liệu Báo cáo khoa học: Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase pdf

Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino

in Mycoplasma pneumoniae, but absence of a gene

encoding a conserved bacterial type I SPase

Ina Catrein, Richard Herrmann, Armin Bosserhoff and Thomas Ruppert

Zentrum fu¨r Molekulare Biologie Heidelberg, Universita¨t Heidelberg, Germany

Mycoplasma pneumoniae is a human pathogenic

bac-terium [1,2], characterized by a small genome of 816

kbp [3], the lack of a bacterial cell wall and a parasitic

lifestyle [4]

Some species of the genus mycoplasma, e.g

Myco-plasma genitalium, Mycoplasma gallisepticum and

Mycoplasma pneumoniae exhibit a flask-like shape,

which is believed to be formed and maintained by a

cytoskeleton-like structure [5–10] This flask-like shape

is caused by the attachment organelle, an asymmetric

extension of the cell composed of an assembly of

unique proteins [11] M pneumoniae interacts with its

host cell by adhering with the attachment organelle to

specific receptors This interaction takes place only if

the P1 protein, the bacterial main adhesin, is inserted

correctly into the attachment organelle [12] The

proper insertion depends, among others, on the

pro-teins P40 and P90 Absence of these propro-teins causes a

random insertion of the P1 protein and a

cytadher-ence-negative phenotype [13] P1 is encoded by

MPN141 and both, P40 and P90 by MPN142 These genes are organized, together with MPN140 which probably encodes a phosphoesterase [14], in the P1 operon [15] In the original publication these genes were called ORF4 (MPN140), ORF5 (MPN141) and ORF6 (MPN142) [15] The names in brackets are the gene names according to a recent reannotation [15,16] MPN142codes for a protein with a molecular mass of

130 kDa, which, however, has been never identified as single protein of the expected molecular mass Instead, two proteins with molecular masses of about 40 kDa (P40) and 90 kDa (P90) had been found in SDS⁄ PAGE and Western blotting experiments [17] An enzyme responsible for the processing of the proposed 130-kDa protein has not yet been identified P40 derives from the N-terminal and P90 from the C-ter-minal part of the predicted 130-kDa precursor protein

It was proposed that P40 and P90 are identical with the proteins B and C missing in certain avirulent mutants [18,19], but so far this has not been proven

Keywords

chemical assisted fragmentation (CAF);

mass spectrometry; protein modification;

signal peptidase

Correspondence

T Ruppert, Zentrum fu¨r Molekulare Biologie

Heidelberg, Universita¨t Heidelberg, Im

Neuenheimer Feld 282, 69120 Heidelberg,

Germany

Fax: +49 6221 545891

Tel: +49 6221 546895

E-mail: t.ruppert@zmbh.uni-heidelberg.de

(Received 15 December 2004, revised

11 March 2005, accepted 7 April 2005)

doi:10.1111/j.1742-4658.2005.04710.x

Although the annotation of the complete genome sequence of Mycoplasma pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we showed experimentally that such an activity must exist in this bacterium, by determining the N-terminus of the N-terminal gene product P40 of MPN142, formerly called ORF6 gene Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino acids 25 and 26 of the P40 precursor protein The experimental results were in agree-ment with the cleavage site predicted by computational methods providing experimental confirmation for these theoretical analyses

Abbreviations

CID, collision induced fragmentation; SPase I, signal peptidase I.

experimentally The P1 protein can be cross-linked

with P40 and P90 under in vivo conditions, indicating

that they form a larger protein complex embedded into

the cytoplasmic membrane [20,21]

To get insight into the function of this protein

com-plex, the subunits had to be characterized in detail

The N-terminal end of P90 was determined by Edman

degradation This protein begins with an arginine at

amino-acid position 455 of the proposed precursor

[22] The predicted molecular mass of P40 is therefore

47 700 This value, however, differs significantly from

the molecular mass of 36–37 kDa seen in SDS⁄ PAGE

This discrepancy could be explained either by

abnor-mal migration of P40 in SDS⁄ PAGE, or more likely,

by additional processing steps causing the observed

reduction in apparent molecular mass

A processing step could take place at the N-terminal

region of P40, as a bacterial signal peptide had been

predicted [23–25] Such signal peptides are normally

cleaved off by a bacterial type I signal peptidase

(SPase) The various type I SPases from

Gram-negat-ive and Gram-positGram-negat-ive bacteria show clear differences

concerning gene size, gene copy number and substrate

specificity, despite the substantial sequence similarities

as indicated by six distinct regions with conserved

amino acids [26] For instance, LepB from Escherichia

coliis 323 amino acids long and exists only as a single

gene copy while the occurrence of multiple type I

SPases within a single species is commonly observed in

Gram-positive bacteria Bacillus subtilis contains five

chromosomally encoded type I SPases named SipS,

SipT, SipU, SipV and SipW, which are only about 200

amino acids in size [26] As M pneumoniae belongs

phylogenetically to the Gram-positive bacteria one

would expect to find a type I SPase similar to the

var-ious sip genes However, no type I SPase typical for

Gram-positive bacteria or for Gram-negative bacteria

(such as LepB) has been identified in M pneumoniae,

although a SPase I activity has been shown to be

essential for cell viability in all bacteria analyzed [26]

To test experimentally whether there is a type I

SPase activity in M pneumoniae, we determined the

N-terminus of P40 as it appears in protein extracts of

M pneumoniae

Determination of the N-terminus of P40 by Edman

degradation failed due to the limited amount of

start-ing material An alternative method is the tryptic

digestion of the purified protein and subsequent

ana-lysis of the derived peptides by mass spectrometry If

a candidate mass is detected and peptide sequencing

proves, that there is no trypsin cleavage site at the

amino terminus of this peptide, as read from the gene

sequence, then this peptide is taken as the N-terminal

peptide of this protein [27] This is, however, not an exact proof, as such a cleavage may also result from proteolytic contaminants of the trypsin being used like chymotrypsin or due to pseudotrypsin formed from trypsin by autolysis [28] These possibilities can be excluded, when the amino terminus of the intact pro-tein is specifically labeled before tryptic digestion Liminga and colleagues [29] introduced an N-hydroxysuccinimide ester of 3-sulfonic-propionic acid (CAF reagent) to modify peptides after tryptic diges-tion for enhanced peptide sequencing using matrix-assisted laser desorption⁄ ionization time of flight mass spectrometry (MALDI-TOF-MS) with chemical assis-ted fragmentation (CAF) [30] We modified this method in a way so that only the N-terminus of the mature P40 was labeled with the CAF reagent After tryptic digestion, the N-terminal sulfonated peptide of P40 could be identified unambiguously

Results

To analyze in more detail the protein complex formed by the proteins P1, P40 and P90, it is a pre-requisite to know the primary structure of the sub-units To get sufficient material for the identification

of the amino terminus, P40 was enriched from cell extracts of M pneumoniae M129 by immunoprecipi-tation with a polyclonal antiserum directed against a P40 fragment corresponding to the sequence from residue 66 to residue 223 [17] The final SDS⁄ poly-acrylamide gel, which was the source of P40 for the N-terminal sequence analysis, is shown (Fig 1) The indicated Coomassie blue stained protein band was recognized by antibodies directed against P40 in western blotting experiments (data not shown) The P40 band was clearly separated from other proteins, but the amount of P40 was not sufficient for sequen-cing by Edman degradation Therefore, we used mass spectrometric analysis of an in-gel digest of P40 To prove that the candidate peptide was the N-terminal peptide of P40, we labeled the N-terminus of P40 within the gel piece before tryptic digestion by

a method described for peptide sequencing by MALDI-TOF MS after chemical assisted fragmenta-tion (CAF) [30]: during the first step of the labeling reaction the e-amino groups of lysine are specifically converted to homoarginine In a second step, the free amino terminal groups of the peptides are sulfonated

by the CAF reagent (Fig 2)

Subsequently, the protein was digested by trypsin and the supernatant was analyzed by ESI-QTOF MS (Fig 3) in the positive ion mode, to get high quality fragmentation pattern for peptide sequencing Fifteen

signals of various m⁄ z-values from the peptide mass

fingerprint were subjected to collision induced

frag-mentation (CID) P40 was identified (probability

based Mowse score of 276) by sequence tags of six

different peptides covering the N-terminal region

(position 26–203) of P40 (Table 1) The fourfold

charged signal with m⁄ z ¼ 850.2 (rmm: 3396.8)

corre-lated with the mass of the peptide 26–55 (calcucorre-lated

rmm: 3260.8), if an increase in the molecular

mass by 136 Da, the mass of the sulfonic acid

modi-fication, is taken into account The identity of this

peptide was confirmed by collision induced

fragmen-tation (CID) The complex fragmenfragmen-tation pattern of

the fourfold protonated peptide shows single, double

and triple charged fragment ions, which can be easily

distinguished from the isotopic pattern After deconvolution, the peptide was identified by the almost complete series of y-fragments and a long series of b-fragments (Fig 4) From the difference of the precursor mass to the y-28 fragment and from the b1 fragment, which both represent the N-terminal part of the peptide, it is evident that the N-terminal amino acid is asparagine (position 26), but increased

in mass by 136 Da Because this modification is only possible at the free a amino group of asparagine, this amino acid must represent the N-terminal amino acid of the protein Interestingly, this peptide can acquire up to four positive charges even when there are only two basic residues present in this sequence

In the peptide mass fingerprint (Fig 3, inset) the unmodified peptide (m⁄ z ¼ 816.18) is also detected, but the signal intensity is only about 20% of that

of the modified peptide Therefore, labeling of P40 within the gel by the CAF reagent occurred in a very efficient way and allowed the identification of the new N-terminus after cleavage of the signal sequence (Fig 5)

Remarkably, the sulfonated peptide 26–55 also appeared with high intensity as sodium and a potas-sium adduct ion, respectively (Fig 3) These salt adducts were most presumably bound by the strong negative charge of the sulfonate group The same observation was found only for one additional peptide

Fig 3 Peptide mass fingerprint of P40 The tryptic digest of P40 was analyzed by ESI-QTOF MS Multiple charged peptide ions cor-responding to P40 are indicated by arrows The inset shows the

m ⁄ z region of the fourfold charged ions of the N-terminal peptide (position 26–54) 816.2 (+ 4) and 850.2 (+ 4) represent the unmodi-fied and the sulfonated peptide, respectively Two additional, four-fold charged peptides with m ⁄ z ¼ 855.67 (rmm: 3418.7) and with

m ⁄ z ¼ 859.66 (rmm: 3434.6) represent the sodium and potassium adduct of the sulfonated peptide 26–55 as evident from the frag-mentation spectra (data not shown).

Fig 2 Labeling of the N-terminus of a protein by the CAF reagent.

In a first reaction lysine residues are specifically converted to

homoarginine by O-methylisourea Then, the free a-amino group at

the N-terminus is sulfonated by the CAF reagent.

1 2 3 kDa

250 150 100 75

50

37

25

15

Fig 1 Enrichment of P40 for mass spectrometric analysis The

pro-teins were separated by SDS ⁄ PAGE (12.5%) Lane 1, molecular

mass marker; lane 2, proteins (1.5 lg) enriched by

immunoprecipita-tion; lane 3, total cell extract of M pneumoniae (7 lg) The gel was

stained with colloidal Coomassie blue The protein band, which was

cut out for mass spectrometric analysis is indicated by an arrow.

with m⁄ z of 436.3, threefold charged It turned out

that this peptide 71–81 (VGDTKLVALVR) contained

a lysine in the middle of the sequence, also modified

by sulfonation (Fig 6)

Other amino acids were hardly, if at all, influenced

by this two step labeling procedure The oxidation of

tryptophan or the conversion of asparagine to aspartic

acid, as observed for peptide 146–152 (ATWVFER)

and peptide 204–226 (VNGVAQDTVHFGSGQESSW

NSQR), respectively, was also found during normal sample preparation [28]

Discussion

Protein identification is greatly facilitated by the growing number of genome sequences To get further insight into the function of a protein, additional infor-mation about post-translational modifications, like

Table 1 The tryptic digest of P40 was analyzed by ESI-QTOF MS Peptide ions of the indicated m ⁄ z-value were fragmented by CID Amino acid sequences were calculated from the fragment ions (letters in bold) or taken from the P40 sequence (letters in italics).

850.2 3396.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26)

855.7 3418.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26), Na + adduct 859.7 3434.6 N(+136)TYLLQDHNTLTPYTPFTTPXDGGXDVVR (26–54) Sulfonated at N (position 26), K + adduct

Fig 4 Identification of the N-terminus of P40 A fourfold charged peptide (m ⁄ z ¼ 850.18) with a molecular mass of 3396.4 Da was fragmen-ted by collision-induced fragmentation, because the calculafragmen-ted mass of peptide 26–54 (rmm: 3260.7) fits well if a CAF modification is assumed (+136 Da) The fragmentation spectrum showed dominant double and triple charged fragment ions To reduce the complexity, the spectrum was deconvoluted and the peptide sequence was obtained from the single charged y-fragment ions (…) and the b-fragment ions (– –) Calculated from the b1 fragment and the y 28 fragment, the N-terminal amino acid consists of asparagine modified by the CAF reagent (+136 Da) Most of the unlabelled fragments in the spectrum are y- and b-ions after neutral loss of H 2 O from the side chains of aspartic acid and threonine.

proteolytic processing steps, are necessary Edman

de-gradation is a widely used technique for determining

the N-terminus of a protein This method, however, is

not only limited by its inability to deal with amino

ter-minal modified proteins, but also by its poor sensitivity

compared to mass spectrometry On the other hand,

mass spectrometry is still not very efficient for

analyz-ing whole proteins despite the progress made duranalyz-ing

the last years Such an analysis normally begins

with the proteolytic degradation of a protein, followed

by the analysis of the cleavage products It is, how-ever, difficult to prove which of the identified peptide represents the amino terminus of the protein To over-come this problem, we labeled the amino terminus of the protein after electrophoretic separation within the excised gel piece

As shown, the N-terminal amino group of P40 could

be sulfonated by the CAF reagent After tryptic diges-tion, the N-terminal sulfonated peptide 26–54 was identified by collision induced dissociation of the four-fold protonated peptide ion The charge state of this peptide is unexpected: a peptide containing two basic amino acids (arginine and histidine) and a blocked amino terminus should not acquire more than two positive charges in positive ion mode On the other hand, the maximal charge state of a peptide can be extended with increasing length which may be due to additional protonation of the peptide backbone in the gas phase under electrospray conditions (T Ruppert, unpublished results) Due to the multiple charged peptide ions formed by electrospray ionization the fragmentation pattern of the N-terminal sulfonated peptide differed significantly from the fragmentation pattern of similar modified peptides using post source decay (PSD) in MALDI-TOF mass spectrometry, where b-ions are not observed [31] The b1 to b5 frag-ments containing the sulfonated N-terminus of the fourfold protonated peptide were detected as positively charged fragment ions This indicated that the strong acid group at the N-terminus was not deprotonated as assumed for MALDI-PSD Therefore, the N-terminal labeling is not only visible from y-ion series but also

by the b-ion series The mass of the b1 fragment cor-responded to an asparagine, increasing in mass by

136 Da Detection of the b1 fragment ion indicates that an amide bond, formed after the reaction the sulfonation reagent, is present at its N-terminus instead of a free amino group [32] Therefore, the asparagine in position 26 was labeled within the intact protein at its a-amino group and represents the amino terminal amino acid of P40 The efficiency of this reaction was quite high, as judged from the signal intensities of the corresponding N-terminal modified peptide, compared to that of the unmodified peptide (Fig 3)

In the peptide mass fingerprint, the N-terminal sulfo-nated peptide 26–54 was detected not only in its pro-tonated form, but also as a sodium and potassium adduct ion (Fig 3) Interestingly, after collision induced dissociation of these peptide ions, only y-frag-ments were observed The sodium and potassium ions, respectively, were most likely bound at the

deproto-Fig 5 SIGNAL P predicted and experimentally verified cleavage site

for P40 The cleavage site for the P40 precursor protein was

predicted by SIGNAL P (Gram-positive network) to be located

between amino acid 25 (alanine) and 26 (asparagine) The values of

the C- (output from cleavage site networks), S- (output from signal

peptide network) and Y-scores (combined cleavage site score) are

shown for each position in the sequence The data were generated

by feeding the first 50 amino acids of the gene products of

MPN142 to the publicly available web server http://www.

cbs.dtu.dk/services/SignalP/ For more details see [24,25] The

experimentally defined N-terminus of the mature P40 agreed with

this prediction (black arrow).

Fig 6 Fragmentation spectrum of the sulfonated peptide 70–80.

The doubly charged peptide ion with m ⁄ z ¼ 653.9 showed intense

sodium and potassium adducts as observed for the sulfonated

N-terminal peptide 26–54 From the fragmentation pattern

(y-frag-ments are indicated by arrow) the peptide was identified as

70-VGDTKLVALVR-80 containing a lysine in the middle of the

sequence, which is probably modified by sulfonation (+136 Da).

nated sulfonic acid group and lost during

fragmenta-tion, which causes neutralization of the b-fragments

The formation of sodium and potassium adducts was

possibly a general property of the sulfonic acid group,

which may help to select such peptides in a complex

mixture after electrospray ionization In fact, we

detec-ted an additional peptide in this tryptic digest, showing

such salt adducts (Fig 3) This peptide contained the

sulfonic acid group in the middle of the sequence

bound to a lysine residue, which was not converted to

homoarginine due to incomplete guanidation Other

amino acids were hardly, if at all, affected by these

two reactions Two amino acid modifications were

detected: tryptophan 248 was oxidized to a minor

extent and asparagine 205 was, to some extent,

con-verted to aspartic acid

We conclude that sulfonation with the CAF reagent

is a simple and efficient procedure to label the amino

terminus of a protein after gel electrophoresis After

in-gel digestion, the identification of a peptide containing

this label at the amino terminus by mass spectrometry

proves that it represents the amino terminus of the

pro-tein Using this method, we showed that asparagine 26

represents the amino terminus of P40 Knowing, that

the first amino acids of the processed P40 and P90 is

the asparagine at position 26 and the arginine at

posi-tion 455 we calculated for P40 a molecular mass of

44.873 kDa As the molecular mass of P40 in protein

extracts, measured by SDS⁄ PAGE is about 36 kDa,

there is an obvious difference in molecular mass of

about 9 kDa between the calculated and the actually

observed masses (Fig 7) It seems reasonable to

assume, that additional proteolytic cleavage took place

Without further information, we can presently not

decide which were the true intermediates in this

pro-cess There could be a premature P40 with an extended

C-terminal region, but a premature P90 with an

exten-ded N-terminal region can not be excluexten-ded (Fig 7)

The best way to analyze this precursor-product rela-tionship would be through puls-chase experiments with radioactively labeled amino acids This approach, however, is hampered by the lack of a defined minimal medium for M pneumoniae

Several predictive methods have been published [23,24], which facilitate the identification of prokaryotic signal peptides A recent study [23] of bacterial and archaeal proteomes proposed that the fraction of puta-tive exported or secreted proteins ranges from 8% (Methanococcus jannaschii) to 37% (M pneumoniae) This means that in M pneumoniae 254 from 688 predic-ted proteins contain a signal peptide Establishing and improving such prediction methods depend on the availability of experimental data Although Nielsen

et al [24] explicitly excluded members of the cell wall-less mollicutes from their analysis of signal peptides, applying their program signal p [25,33] precisely pre-dicted the cleavage site for P40 (Fig 5) This result was confirmed by the program exprot [23] Both methods failed to confirm the cleavage site of the main adhesine P1 of M pneumoniae This is the only other M pneu-moniae protein, of which the N-terminus of the mature protein was experimentally determined For this pro-tein, the predicted cleavage site [23,24] between amino acids 27 and 28 disagreed with the experimental data, which showed that the processed P1 protein begins with amino acid 70 of the precursor protein [25,34,35] Although a multivariate data analysis indicated that signal peptides of the mollicutes differ significantly from

E coliand Gram-positive bacteria [36], a signal peptide

of 70 amino acids is not in agreement with this multi-variate data analysis Therefore, the simplest explan-ation would be that the N-terminus of the mature P1 is generated by an additional processing step

The most interesting question concerns the signal peptidase activity observed in M pneumoniae [26], because a conserved bacterial type I signal peptidase has not been found in annotations of complete genome sequences, neither in M pneumoniae [3,16] nor in the phylogenetically closely related Mycoplasma genitalium [37] They were, however, found in Mycoplasma galli-septicum [38] and Mycoplasma pulmonis [39] In con-trast, the type II signal peptidase, that is specific for signal peptides from lipoproteins of the murein lipo-protein type of E coli [40], occurs in all mollicute spe-cies sequenced so far As the results of our analysis of the gene products of MPN142 proved that a SPase I like activity is present in M pneumoniae, a correspond-ing, hitherto unidentified gene, has to code for it In the course of the re-annotation of the M pneumoniae genome sequence the two genes MPN032 and MPN294 were proposed as possible candidates

enco-Fig 7 Schematic model for processing of the gene product of

MPN 142 Cleavage into P40 and P90 takes place after amino acid

454 The N-terminus of the mature P40 starts with amino acid 26.

The molecular mass of about 36 000 Da of P40, as determined by

SDS ⁄ PAGE would correspond to a protein reaching from amino

acid 26–365 (P40 340 ) According to this scheme a premature P40

(amino acid 26–454) could be an intermediate as well as a

pre-mature P90 (amino acid 366–1218).

ding a SPase I like activity [16] Using recombinant

P40 preprotein or suitable synthetic peptide

sub-strates [26] subfractions of protein extracts from

M pneumoniae can now be assayed for signal

pepti-dase I activity facilitating the identification of the

corresponding gene⁄ protein

Experimental procedures

Bacteria

M pneumoniae reference strains M129 (ATCC 29342;

type 1; broth passage no 31) and FH (ATCC 15531;

sub-type 2 [41]; broth passage no 5) were cultivated as

described previously [42] and stored at)80
C For sample

preparation, M pneumoniae cells were grown adherently at

37
C in 100 mL of modified Hayflick medium [43] in

150 cm2cell culture flasks (Greiner, Flacht, Germany)

Enrichment of IgG

The IgG fraction of 2 mL rabbit anti-FP130K-1 serum (no

42328 [17]), was precipitated with Na2SO4 by mixing the

serum with 2 mL phosphate buffer (0.1 m KH2PO4, 0.1 m

Na2HPO4, pH 7.4) and adding 4 mL of a Na2SO4solution

(34%, w⁄ v) The suspension was incubated for 5 min at

20
C and then centrifuged at 20
C for 5 min at 12 000 g

The supernatant was discarded, the pellet dissolved in 2 mL

phosphate buffer and the Na2SO4 precipitation repeated

twice The final pellet was dissolved in 2 mL phosphate

buf-fer The concentration of this suspension was determined

by reading the absorbance at 280 nm and using an

extinc-tion coefficient of 1.4 for a 1 mgÆmL)1solution of IgG We

obtained from 2 mL of serum about 10 mg protein, mainly

IgG as revealed by SDS⁄ PAGE The IgG-enriched fraction

retained the ability to recognize P40 in western blotting

experiments (I Catrein, unpublished results) SDS⁄ PAGE

and western blotting were performed as published recently

[44]

Cross-linking of IgG to magnetic beads

The enriched IgG fraction (10 mg) was bound to 2 mL

sus-pension of magnetic beads, which carried recombinant

Pro-tein A covalently attached (Dynabeads ProPro-tein A, Dynal

Biotech, Oslo, Norway) following the instructions of the

manufacturer

Isolation of P40 from cell extracts

M pneumoniae M129 from 10 cell culture flasks (150 cm2,

100 mL modified Hayflick medium) were collected, washed

twice with phosphate buffer and the pellet suspended in

2 mL lysis buffer (500 mm NaCl, 50 mm Tris⁄ HCl pH 7.5,

0.5% Triton X-100 and the protease inhibitor cocktail com-plete, EDTA-free (according to the manufactures recom-mendation; Roche, Basel, Switzerland) The bacteria were sonicated with a Branson sonifier for 8· 15 s at 4
C with intervals of 1 min and the suspension separated in pellet and supernatant by centrifugation (60 min, 75 000 g) in a Beckman TL100 ultracentrifuge The protein concentration

of the 2 mL supernatant was about 25 mgÆmL)1 as meas-ured with the Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as standard It contained almost all of the P40 protein as revealed by western blotting For the isolation of P40, the

2 mL supernatant were incubated with the prepared mag-netic beads (see previous paragraph) overnight at 4
C with gentle agitation After magnetic separation, the beads were washed 6 times with 6 mL of 0.1 m sodium phosphate buf-fer, pH 8,1 P40 was eluted by adding 540 lL 0.1 m citrate buffer, pH 2,8 to the beads and incubating the suspension for 2 min at 20
C The beads were again removed by the magnet and the supernatant containing now the P40 was neutralized by adding 1 mL of 0.1 m sodium phosphate buffer, pH 8,1 This protein solution was concentrated with

a spin column with a 10 kDa cut-off (Vivaspin concen-trator, Vivascience, Hannover, Germany) to a volume of

24 lL It contained enough P40 for separating the P40 protein by SDS⁄ PAGE and characterizing it by mass spectrometry

Protein determination and mass spectrometry

The Coomassie blue stained band containing P40 was excised from the gel After washing, the gel piece was trea-ted with 30 lL 10 mm DTT at 60
C for 10 min and alkyl-ated with 30 lL 40 mm iodacetamide at room temperature for 15 min The gel piece was again washed with 25 mm ammonium bicarbonate, shrunk in acetonitrile and incuba-ted with water for 15 min After removal of excess water,

100 lL of O-methylisourea hemisulfate (140 mm in 200 mm sodium bicarbonate, pH 10) was added and incubated over night at room temperature The supernatant was removed, the gel piece washed twice with water and shrunk in aceto-nitrile on ice After removal of acetoaceto-nitrile, 6 mg CAF rea-gent (chemical assisted fragmentation reagent; kindly provided by Amersham Biosciences, Freiburg, Germany) (Fig 1) dissolved in 60 lL 250 mm sodium bicarbonate,

pH 9.4, was added After 10 min on ice the Eppendorf tube was quickly brought to room temperature for additional

5 min Then, the reaction was stopped by addition of 2 lL 50% hydroxylamine solution After 1 h the supernatant was removed and the gel piece washed twice with water,

25 mm ammonium bicarbonate, pH 8.5 After shrinkage in acetonitrile, the gel piece was incubated with 15 lL 25 mm ammonium bicarbonate, pH 8.5 containing 140 ng modified porcine trypsin (8 ngÆlL)1) (Promega, Madison, WI, USA) for 4 h at 37
C Digestion was stopped by formic acid

(final concentration: 2%) and the sample was stored at

)20
C MS analysis was performed on a Q-TOF mass

spectrometer (Applied Biosystems, Darmstadt, Germany)

equipped with a nano-ESI ion source (Protana, Odense,

Denmark) as described previously [27] If indicated,

MS⁄ MS spectra were deconvoluted using Bayesian Peptide

Reconstruct (mass tolerance: 0.1 Da; S⁄ N threshold: 2)

pro-vided with the analyst qs software (Applied Biosystems)

Acknowledgements

We thank E Pirkl and M Ellis for skillful technical

assistance, C.-U Zimmerman for critically reading the

manuscript, M T Saleh for providing the latest results

of a signal peptide analysis of M pneumoniae and the

Deutsche Forschungsgemeinschaft for financial

sup-port (He 780⁄ 12–2 and SFB 382)

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Supplementary material

The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4710/EJB4710sm.htm

Fig S1 (A) Isolation of P40 from cell extracts, (B) isolation of P40 from cell extracts as revealed by wes-tern blot and (C) identification of the N-terminus of P40

Table S1 Peak list of the fragmentation spectrum of the fourfold charged peptide with m/z ¼ 850.18