Download C.N.R. Short-term Mobility Program 2008 Gabriella Pocsfalvi

C.N.R. Short-term Mobility Program 2008
Gabriella Pocsfalvi
Proteomic analysis of Sulfolobus solfataricus flagellum
Introduction
In this project in collaboration with Biomedical Analysis Department, Institute of Medical Physics
and Biophysics (IMPB), University of Munster (Germany) we aim to study the motility organelle of
the aerobic thermoacidophilic crenarchaeone Sulfolobus solfataricus, a model organism of
hyperthermophilic archaea that grows optimally at 80°C. Most of the archaea have small flagellar
filaments (10-14 nm diameter) which are primary responsible for motion but they supposed to have
fundamental role also in cell-cell communication, signaling, adherence, nutrient and extracellular
DNA uptake as well. Nonetheless flagellation in archaea is widespread, structural and functional
characterization of archaeal flagella today is still far less comprehensive than bacterial flagella or
pili. The fla operon of S. solfataricus encodes seven open reading frames: five putative accessory
proteins (G, F, H, I, J), one structural protein (flaB) and one ORF (SSO2322) of which homolog can
only be found in Sulfolobus genomes. S. solfataricus carries only one copy of the flagellin gene and
does not show reversed swimming or tumbling. Therefore, it is supposed that the structure of
flagella from this organism should be homogenous, in contrast to polymorphic flagella from other
archaeal species. Images of flagellar filaments isolated from S. solfataricus have recently been
acquired by transmission electron microscopy; and transcriptional regulation of flaB genes in
different nutrient conditions has also been reported (Szabo et al., J. Bacterol, 189, 4305, 2006).
Archaeal flagellar proteins are secreted via a dedicated flagellar secretion system. Flagellin protein
of S. solfataricus have, for example, an unusual “type IV pilin-like” signal peptide which is cleaved
by the archaeal homologue (PibD, preflagellin peptidase) of the type IV pilin signal peptidase.
Interestingly, the IV pilin-like signal sequences are not only found in flagellin protein but also in the
precursors of other 15 extracellular proteins, like the solute (sugar) binding components of ABC
transporters. Isolation, structural and functional characterization of the native protein products of fla
operon expressed by S. solfataricus, and especially the flagellar bindosome (i.e. all the protein
interacting with flagellum and isolated with it), however, are still missing. Therefore, the principal
aim of this project was to apply mass spectrometry based proteomics for the exploration of the
protein composition of flagellar fraction isolated from S. solfataricus.
Preparation of flagella fraction from S. solfataricus P2 for proteomic analysis
S. solfataricus P2 grown at 80 °C and pH 3.2 in rich medium (TYS) up to the middle exponentially
phase (in 36h). Flagellar filaments were isolated both from the sheared cells (S) and from the
culture supernatant (E) by CsCl gradient ultracentrifugation using a modified method of Kalmokoff
(J. Bacterol., 1988, 170, 1752-1758). Shearing were performed twice resulting in samples S1 and
S2, respectively (Figure 1 a). From the three samples (S1 and S2 sheared and E3 obtained from the
culture supernatant) the following five fractions were collected after CsCl gradient centrifugation
(Figure 1.a):
S1
Flag_SSO_Sheared1
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C.N.R. Short-term Mobility Program 2008
S2a
Flag_SSO_Sheared2a (upper band)
S2b
Flag_SSO_Sheared2b (lower band)
E3a
Flag_SSO_Exo3a (upper band)
E3b
Flag_SSO_Exo3b (lower band)
Gabriella Pocsfalvi
Protein concentrations were measured by the Fluoroprofile kit (Sigma) and 8 µg of each fraction
were loaded on sodium dodecyl sulphate-ployacrylamide gel electrophoresis (SDS-PAGE) (Figure
1.b).
1D-SDS-PAGE based proteomics
1D-SDS-PAGE separation and peptide extraction: Sample (8 μg) was solved in 20 μL buffer (0.125
M Tris-HCl pH 6.8, 4% SDS, 20% Glycerol, 0.2 M DTT, 0.02% bromophenol blue), boiled at 100
°C for 5 min and loaded on 12% polyacrylamide gel according to Laemmli. Selected gel bands were
cut manually and in-gel digestion was performed according to Shevchenko using 30 μL 10 mM
DTT in 100 mM NH4HCO3 for reduction, 30 μL 55 mM IAA in 100 mM NH4HCO3 for alkylation
and 30 μL trypsin and/or chymotrypsin at 6 ng/μL concentration in 5% acetonitrile and 25 mM
NH4HCO3 for enzymatic digestion. Samples (10 μL) were analyzed by nanoflow-HPLC-ESIMS/MS.
Figure 1. (a) S. solfataricus flagellar samples separated by ultracentrifugation from sheared cells
(S1 and S2) and from the extracellular medium (E3). (b) SDS-PAGE of samples obtained after
ultracentrifugation: S1, S2a, S2b, E3a and E3b. Gel bands between 27-34.6 kDa were selected for
nano-HPLC-ESI-MS/MS IDA analysis.
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C.N.R. Short-term Mobility Program 2008
Gabriella Pocsfalvi
Nano-HPLC-ESI-MS/MS analysis: Tryptic and chymotryptic peptides were analyzed by a hybrid
quadrupole time-of-flight (QTOF) instrument, QSTAR Elite (Applied Biosystems, Foster City,
CA/Toronto Canada) equipped with a nanoflow electrospray ion source. Pulled silica capillary (360
OD/100 ID, Tip 30 ID) was used as nanoflow tip. Samples (1-10 μL out of 40 μL) was loaded,
purified and concentrated on a pre-column PepMap, C18, 5mm length, 300Å, (LCPackings,
Sunnyvale, CA USA) at 30 μL/min flow rate. Peptide separation was performed on a capillary
column, PepMap, C18, 15cm length, 75μm ID, 300Å (LCPackings, Sunnyvale, CA USA) using
solvents A) 2% acetonitrile in 0.1% formic acid (HCOOH) and 0.025% trifluoroacetic acid (TFA)
and B) 98% acetonitrile in 0.1% HCOOH and 0.025% TFA at flow rate 300 nL/min by Ultimate
3000 (Dionex, Sunnyvale, CA USA). The following gradient was used: 5-50% B in 30 min, 5098% B in 6 sec. CID experiments were carried out in information dependent analysis (IDA) mode
using the Analyst QS 2.0 software. Nitrogen was used as collision gas. Database search was
performed by Mascot Server 2.2 program (Matrix Science Ltd, London UK) using the following
criteria: database: NCBInr (03-Apr-2008), taxonomy: Sulfolobus solfataricus, type of search:
MS/MS Ion Search, enzyme: trypsin with one missed cleavage site allowed, fixed modifications:
carbamidomethyl, variable modifications: oxidation on methionie, automatic error tolerant search;
peptide tolerance 50 ppm, MS/MS tolerance 0.08 Da.
SELDI-TOFMS: SELDI-TOF mass spectra were obtained using PCS-4000 (BioRad) linear LDI
instrument on the complex protein samples without separation and prior enzymatic digestion. NP20
protein chips (BioRad) was used and sinapinic acid was applied as matrix.
RESULTS
SDS-PAGE bands present in the mass range between mass markers at 27 and 34.6 kDa (Figure 1 b)
were cut from sample S1, S2a, E3a and E3b, and were analyzed in information dependent analysis
mode by nano-HPLC-ESI-MS/MS. Identified proteins are listed in Table 1. Among these proteins
we could not identified the putative flagellin B protein (SSO2323) (Figure 2).
LAGLDTAIILIAFIITASVLAYVAINMGLFVTQKAKSTINKGEETASTALT
LSGSVLYAVNYPLNTRSYWIYFTVSPSSGVSSVELSPTTTAISFTASAEG
VTYSNIYKYTLLTVSPSELANVVYANGQYLDLVNQQTSAGQTYVYYP
NPYYALLALNYTLYNYYLSTKTPSPIFINSSILSLSSLPSWLKNDNSFTFT
LNISGKLVTYYVFVNQTFAFTYPVAGDPLIGSAIAPAGSVIGVILLFGPD
LGSHVFQYQTITIQITPNIGSPLTISEYIYQPEGSVSVIG
Figure 2. Amino acid sequence of processed putative flagellin B protein (SSO2323)
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C.N.R. Short-term Mobility Program 2008
Gabriella Pocsfalvi
In sample S1 three hypothetical protein have been identified together with a peptidase related
protein (SSO2181). The most abundant protein in sample S1 is SSO0881 (Mw. 25020Da) a
conserved hypothetical protein similar to yeast and mammalian VPS24, which has been implicated
in cell sorting and trafficking. Another putative non characterized protein in the same sample is
SSO2749. This protein shows conserved domain of Linocin_M18 bacteriocin protein. Many Grampositive bacteria produce antimicrobial peptides, generally termed bacteriocins. These polypeptides
usually has less than 50 amino acid residues long cationic, contain an amphiphilic or hydrophobic
region, and often kill their target cells by permeabilising the cell membrane. Antimicrobial peptides
with these characteristics are also produced by plants and a wide variety of animals, including
humans, and are thus widely distributed in nature. The Linocin_M18 region is found mostly in
eubacteria, though homologous sequences have been identified in archaea as well. The best-scoring
hit on the sequence of SSO2749 was member pfam04454. The third hypothetical protein, SSO0389
is a TPA surface layer glycoprotein based on homology. In sample S2 subunits of two large protein
complexes, thermosome and proteosome were found. Thermosome of Sulfolobus solfataricus, a
type II chaperonin composed of eight alpha and eight beta subunits, has been shown to be an RNAbinding protein that participates in ribosomal RNA processing. Proteasomes, on the other hand, are
large, organelle-like structure. Their proteolytic active sites are compartmentalized within a central
chamber formed by rings of proteins stacked into a cylindrical particle. This is common to energydependent proteases and is postulated to minimize cellular damage as well as facilitate hydrolysis of
substrate proteins. Proteasome and thermosome are believe to be compartmentalized in the cytosol,
therefore, we have concluded that sample S2 were contaminated due to cell lyses occurred sample
defrost prior cell shearing. To remove contamination sample S2 was further purified by CsCl
gradient ultracentrifugation.
The most abundant protein identified in sample E3 obtained from the culture broth, similarly to
sample S1, was SSO0881 hypothetical protein. Amongst other hypothetical proteins, membrane
transporter, ABC transporter, ATP binding component, transposons were also identified, and there
role in the bindosome of S. solfataricus will be the subject of further studies.
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C.N.R. Short-term Mobility Program 2008
Gabriella Pocsfalvi
Table 1. Proteins identified in samples S1, S2 and E3 by SDS-PAGE nano-HPLC-ESI-MS/MS.
Sample
S1
S2a
1DE
band
1
2
6
7
E3a
1
E3b
2
1
2
3
Locus
tag
SSO0881
SSO2749
SSO0389
SSO2181
SSO0282
SSO0862
SSO0216
SSO0405
SSO0732
SSO0356
SSO0407
SSO0768
SSO3254
SSO0421
SO0282
SSO0758
SSO0407
SSO0862
SSO073
SSO0286
SSO0345
SSO2450
SSO0736
SSO0216
SSO1514
SSO094
SSO0421
SSO1457
SSO0698
SSO0732
SSO0881
SSO0451
SSO1960
SSO0881
SSO0881
SSO0451
SSO2734
SSO0200
SSO3250
SSO3079
SSO2241
SSO0167
Acc. Num.
NCBInr
gi|15897772
gi|13816077
gi|13813536
gi|13815479
gi|13813422
gi|13814044
gi|13813351
gi|13813558
gi|13813902
gi|13813501
gi|13813560
gi|13813941
gi|13813362
gi|13813572
gi|13813422
gi|13813928
gi|13813560
gi|13814044
gi|13813906
gi|13813429
gi|13813486
gi|13815753
gi|13813904
gi|13813351
gi|13814743
gi|13814120
gi|13813572
gi|13814682
gi|13813867
gi|13813902
gi|15897772
gi|15897382
gi|15898756
gi|15897772
gi|15897772
gi|15897382
gi|13816059
gi|13813336
gi|13816696
gi|13816494
gi|13815540
gi|13813298
SSO0079
SSO0210
SSO0512
SSO1845
gi|13813212
gi|13813345
gi|13813671
gi|13815103
gi|13814462
gi|13816544
gi|13816671
gi|13814089
gi|13815479
SSO3123
SSO1266
SSO0909
SSO2181
Name
Score
Mw
Mascot
hypothetical protein
Conserved hypothetical protein
Hypothetical protein
Peptidase related protein
Thermosome beta subunit
Thermosome alpha subunit
elongation factor 1-alpha
PCNA
Conserved hypotetical protein
Phosphate regulatory protein, putative
metE-2
Activator 1, replication factor C, small subunit
DNA-directed RNA polymerase, subunit B''
AAA family ATPase
Thermosome beta subunit
Peptidyl-prolyl cis-trans isomerase, FKBP-type rotamase
metE
Thermosome alpha subunit
Proteasome subunit
Conserved hypothetical protein
LSU ribosomal protein L1AB
TATA binding protein (TBP)-interacting protein, putative
Conserved hypothetical protein
Elongation factor 1-alpha
Conserved hypothetical protein
Fibrillarin-like pre-rRNA processing protein
AAA family ATPase
NAD specific glutamate dehydrogenase
SSU ribosomal protein S5AB
Conserved hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
Conserved hypothetical protein
Membrane transporter
BPS2 protein homolog (bps2)
Methyl coenzyme M reductase system, component A2
homolog
Bacterial-like DNA primase (dnaG)
DNA/pantothenate metabolism flavoprotein (dfp)
First ORF in transposon ISC1217
First ORF in transposon ISC1217
ABC transporter, ATP binding protein
Hypothetical protein
Conserved hypothetical protein
AAA family ATPase, p60 katanin
Peptidase related protein
347
83
46
31
316
189
101
88
76
34
33
32
29
25
418
180
142
120
97
89
79
75
63
57
50
47
46
44
39
34
749
178
44
304
329
153
32
201
42
39
36
33
25020
29523
131899
117402
60387
59695
48573
29333
30232
40360
38340
37774
54173
86578
60387
26700
38340
59695
26639
42681
24512
53283
28095
48573
29968
26520
86578
45538
24200
30232
25020
31896
31687
25020
25020
31896
11874
11084
27496
46490
68610
42541
32
32
32
32
30
29
28
143
25
45382
45892
27584
22942
32315
51345
17612
42278
117402
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C.N.R. Short-term Mobility Program 2008
Gabriella Pocsfalvi
Intact protein analysis in samples S1 and the re-purified S2 by SELDI-TOFMS
SELDI-TOFMS was used to analyze samples S1 and S2 after repeated CsCl gradient
ultracentrifugation. Figure 3 shows the protein pattern of sample S1. The most abundant protein
component of this sample has a molecular mass 24804 Da. This is in agreement with the nanoHPLC-ESI-MS/MS results, and due to the hypothetical protein identified as SSO0881. Sample S2
after repeated step of purification has a similar pattern as S1 (Figure 4). In addition to the high
intensity peak at m/z 24908, one can observe a couple of lower intensity peaks at 30-31 kDa as well.
These peaks could correspond to structural flagellin proteins (Mw. 31171 Da). Further analyses of
this sample, in addition to the newly prepared flagellin samples are in progress.
20000
30000
40000
50000
60000
24904.00
15
uA
10
1050167151_spotB_2.6
5
23288.75
0
20000
30000
40000
50000
60000
Figure 3. SELDI-TOF mass spectrum of Sample S1 on NP20 proteinchip.
20000
30000
40000
50000
60000
24908.44
30
uA
20
1050167151_spotD_3.6
23290.44
10
19992.04
26389.65
31273.82
30136.69
49919.74
0
20000
30000
40000
50000
60000
Figure 4. SELDI-TOF mass spectrum of Sample S2 (S2a and S2b) after repeated step of purification and
acquired on NP20 proteinchip.
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