Supplemental Material and Methods
2.1 Research specimens
Live specimens of Cryptochiton stelleri were collected from Monterey, California
5
between October 2009 and March 2011 and maintained live in a recirculating seawater
system until ready for use. The radular teeth were dissected from these specimens and rinsed
in de-ionized water to remove any loose organic debris for further analysis. All biological
samples were collected, handled, and shipped in strict compliance with all CITES rules and
regulations.
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2.2 TEM analysis of partially mineralized radular teeth
The extracted radular teeth were immediately dehydrated through a graded series of
ethanol treatments. Following complete dehydration to 100% ethanol, a section of the radula
containing the partially mineralized teeth （row 15 in Figure 1B）was removed and fixed with
2.5% gluteraldehyde at room temperature followed by post fixation with 1% osmium
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tetroxide at 4 °C. After embedding the sample in Spurrs resin, resin blocks were cut into 80
nm thick, thin section by a microtome and stained with 2.6 % lead citrate and 2.0 % uranyl
acetate. The sample was analyzed with a Tecnai T12 Transmission Electron Microscope at
120 kV.
2.3 Protein extraction from the radular teeth
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A section of the radula containing fully mineralized radular teeth was excised from
the rest of the radula and was subsequently dried in a lyophilizer. The bases and cusps of
these mature teeth were physically separated from the radular membrane (See the structure of
radular tooth in figure 1A) and then from each other using forceps. Both parts (i.e., bases and
cusps) were washed thoroughly with de-ionized water, 70% ethanol and subsequently
5
re-dried. The cusps (containing the magnetite and iron phosphate) were then ground into a
fine powder using an agate mortar and pestle. To isolate the cusp-associated proteins, the
ground cusps were treated with 1% sodium dodecyl sulfate (SDS) solution for 30 minutes at
100 °C. The proteins were also extracted from the membrane plus base fraction using same
procedure. The concentrations of extracted proteins were quantified by the bicinchoninic acid
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(BCA) assay using Bovine serum albumin (BSA) as a standard.
2.4 SDS-PAGE and amino acid sequencing
Tricine/SDS-PAGE was performed under denaturing conditions according to the
method of Schägger and von Jagow [1]. The gel was fixed and protein spots were visualized
using SilverQuest Silver Staining Kit (Invitrogen, CA). For amino acid sequencing, the
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proteins were separated by SDS-PAGE as described above and stained with colloidal
Coomassie G-250, followed by electroblotting to a PVDF membrane (Millipore, Bedford,
MA, USA). The proteins blotted onto PVDF membrane were then analyzed by edman
sequencing performed at the PAN facility at Stanford University (Stanford, CA, USA).
2.5 LC-MS/MS analysis
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For mass spectrometric analysis, 40 μg of membrane plus base and cusp proteins
were separated on a Tricine/SDS-PAGE gel at a constant potential (i.e., 125 V) for 15 min.
The gel was stained overnight with a Colloidal Blue (Invitrogen, CA) suspension. After
staining, each lane was cut into one slice and digested with trypsin following the protocol of
Shevchenko et al. [2]. The in-gel digests of individual gel lanes were analyzed by a Waters
5
system of 2D nano-Acquity UPLC (ultra performance liquid chromatography) and Q-TOF
Premier tandem mass spectrometer (Waters, Milford, MA). The membrane plus base and
cusp samples were analyzed pair - wise so that system variation would be minimized.
A combination of high pH reverse phase and conventional low pH reverse phase
served as a MudPIT method following manufacturer’s recommendation (Waters, Milford,
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MA). The solvents for the first dimension LC of high pH reverse phase was 20 mM
ammonium formate, pH10 (solvent A) and 100% acetonitrile (solvent B), respectively. The
online fractionation was carried out with a trap column and percentage of solvent B. The
peptide sample was loaded onto an XBridge BEH130 C18 trap column (8 µm particle, 300
µm i.d., 5 cm long, PN# 186003682, Waters, Milford, MA). The partial-loop sample loading
15
method was configured with a 10 µl sample loop in the auto sampler for maximal loading
with zero sample loss (6 µl for each experimental injection). The fractionation and desalting
was carried out using a 2D-dilution method configured within the 2D nano-Acquity and
controlled with MassLynx 4.1 (Waters, Milford, MA). There were 10 fractions and 2 flushes
based on percentage of solvent B as follows: 1. 8.2%; 2. 11.7%; 3. 13%; 4. 14.5%; 5. 15.9%;
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6. 17.4%; 7. 18.9%; 8. 20.4%; 9. 23.6%; 10. 45%; 11. 65% (flush 1); 12. 80% (flush 2). Mass
spectrometry data were acquired for all of the 12 fractions.
For the second dimension LC, a BEH130 C18 column (1,7 µm particle, 75 µm i.d.,
20 cm long, PN# 186003544, Waters) was used for peptide separation. A Symmetry C18 (5
µm particle, 180 µm i.d., 20 mm long, PN# 186003514, Waters) served as a trap/guard
5
column for desalting and preconcentrating the peptides for each MudPIT fraction. The
solvent components for peptide separation were as follows: mobile phase A was 0.2% formic
acid in water, and mobile phase B was 0.2% formic acid in acetonitrile. The separation
gradient was as follows: 0 to 1 min, 0% B; at 3 min, 3% B; at 5 min, 8% B; at 70 min, 45%
B; at 85 min, 60% B; at 95 min, 90% B; at 95 to 100 min, 90% B; at 105 min, 10% B; at 110
10
min, 0% B; at 110 to 135 min, 0% B. The nano-flow rate was set at 0.3 µl/min without
flow-splitting. A blank injection using the same LC method was introduced between each
sample to remove carry-overs or contaminants.
The parameters for the MS instrument were following: nanoESI+ selected as
ionization source using the Nano-sprayer (Waters) coupled with a PicoTips emitter (PN#
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FS360-20-10-CE-20,New Objective, Woburn, MA), capillary voltage set at 3 kV, sample
cone voltage 42 V, extraction cone voltage 3.0 V, collision energy for MS1 scan 5 V, TOF
reflectron operated in V-mode, source temperature set at 100 °C, MS resolution was 10,000 at
FWHM
for V-mode, MCP/TDC detector (micro-channel plate/time-to-digital converter)
voltage set at 1950 V. Mass accuracy was externally calibrated using a standard peptide such
20
as GFP, and all MS measurement was within 123 ppm or better depending on room
temperature and day-to-day variance. Both LC and MS operation and data acquisition were
controlled by the central software, MassLynx 4.1 (Waters).
Nano-LC/MS/MS was carried out for each fraction of MudPIT experiment with a
DDA (data-dependent acquisition) survey method, and data was acquired between 15 and 115
5
min of LC run time with MS1 m/z range from 350 to 1900. MS1 scan time for precursor ions
was 1 sec with 0.1 sec inter scan delay. For each MS1 scan, maximum of four ions were
selected (if and when individual peak intensity was above 30 counts/scan) for MS/MS scan
with the CID (collision induced dissociation) method to generate fragments spectrum.
Individual MS/MS scans switched when TIC reached at 2000 counts or total scanning time
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exceeded 8 sec. Single MS/MS scan time was set for 1.9 sec with 0.1 sec inter scan delay. A
quadrupole collision cell was used for CID fragmentation, and argon gas was chosen as
collision gas with flow rate 0.45 mL/min. A charge state recognition file was used to
determine collision energy based on m/z value and charge state of each ion. Deisotoping
method was employed to avoid repetitive scans for the same peptide ion with deisotoping
15
window set at 3 Da. Real time exclusion was enabled with exclusion window set at 80 mDa
for 10 min. Only ions with multiple charges were selected for MS/MS scan.
2.6 Data analysis
All MS/MS spectra were then processed with the PLGS2 program (Waters, Milford,
MA, USA) to generate PKL text files. Since the genome of Cryptochiton stelleri was
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unsequenced, the pkl files were searched against an Acanthopleura hirtosa expressed
sequence tag (EST) database (from the National Center for Biotechnology Information
(NCBI), containing 498 entries) and non-redundant NCBI (NCBInr) protein database with the
MASCOT algorithm for protein identification. The following parameters were used for
MASCOT searches: monoisotopic mass, peptide mass tolerance 120 ppm, MS/MS tolerance
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0.2 Da, peptide charge 5+, missed cleavages 1, oxidation of methionine, histidine, tryptophan,
N-terminal pyroglutamylation, N-terminal, Lysine acetylation and N-terminal formylation, as
variable modifications. The results with peptide ion scores more than 70 (NCBInr) and 22
(Acanthopleura hirtosa EST), (p<0.05) were considered to be significant. MASCOT searches
were also performed against the decoy database under same condition to calculate the false
10
discovery rate. For all analyses, false discovery rates were 0% for peptide matches above
identity thresholds in MASCOT search. PEAKS Client 5.2 (Bioinformatics Solutions Inc.,
Waterloo, Ontario, Canada) was used to derive de novo peptide sequences from unassigned
spectra. The same parameters were used to generate peptide sequences as the ones used in
MASCOT. The peptide sequences obtained from de novo sequencing were used for a
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SPIDER (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) homology search
against an Acanthopleura hirtosa EST database and MS-BLAST search against NCBI
non-redundant protein database. A SPIDER hit was defined as significant if the SPIDER
score was more than 90% [3]. A MS-BLAST hit was defined as significant if the score of the
High Scoring Pair (HSP) was higher than the threshold value as described previously [4]. The
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identified ESTs annotations were determined via the BLASTP search against the NCBI
non-redundant protein database. Furthermore, the spectra of trypsin, keratin and other
contaminants were hand picked from the data. The identified proteins carrying the same
annotations derived from different organisms were counted as one protein according to the
previous studies using the cross-species protein identification method [5].
5
The peptides predicted from PEAKS which were unassigned to proteins were further
analyzed as followings. In the analysis, de novo peptide sequences harboring amino acids
with a confidence level more than 60% and consisting of more than 7 amino acids were used.
The peptide sequences from cusp fraction that meet above condition were compared with that
from membrane and base fraction and the resulting cusp fraction - specific peptides were
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listed in Supplementary Table S3. BLASTP searches of de novo peptide sequences were
performed on the NCBI non-redundant protein database, an Acanthopleura hirtosa EST
database and the mollusk-restricted EST database using the PAM30 scoring matrix. The
MS/MS spectra of de novo peptide sequences used for protein identification were manually
verified (Supplementary Figure S1).
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The MS data used for protein identification were deposited in the Proteomics
Identifications
Database
(PRIDE:
www.ebi.ac.uk/pride),
under
accession
numbers
22459-22470.
2.7 Replicates
The protein identification were based on two technical replicates. The proteins
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identified as cusp fraction – specific in both technical replicates were listed in Table 1. For
each of these two replicates, several biological replicates (see table below) have been
analysed.
Technical
replicates
Biological
replicates
1st analysis
Radular
teeth #1-10
No. of proteins
identified from
cusp fraction
Radular
teeth #11-22
2nd analysis
1st analysis
2
7
No. of cusp fraction
specific proteins
No. of cusp
fraction specific
proteins
No. of cusp
fraction specific
peptides
12
6
8
455
16
5
13
149
2nd analysis
6
No. of proteins
identified from
membrane +base
fraction
1st analysis
446
2nd analysis
9
140
No. of cusp fraction
specific peptides
References
5
[1] Schagger, H., von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel
electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem
1987, 166, 368-379.
[2] Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Mass spectrometric sequencing of
proteins silver-stained polyacrylamide gels. Anal Chem 1996, 68, 850-858.
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[3] Wang, Q., Han, H., Xue, Y., Qian, Z., et al., Exploring membrane and cytoplasm
proteomic responses of Alkalimonas amylolytica N10 to different external pHs with
combination strategy of de novo peptide sequencing. Proteomics 2009, 9, 1254-1273.
[4] Shevchenko, A., Sunyaev, S., Loboda, A., Shevchenko, A., et al., Charting the proteomes
of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass
spectrometry and BLAST homology searching. Anal Chem 2001, 73, 1917-1926.
[5] Coumans, J. V., Moens, P. D., Poljak, A., Al-Jaaidi, S., et al., Plant-extract-induced
changes in the proteome of the soil-borne pathogenic fungus Thielaviopsis basicola.
5
Proteomics, 10, 1573-1591.
Supplementary Figure Legends
Figure S1 MS/MS spectra of single peptide identification and MS/MS spectra used for de
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novo sequencing.
Figure S2 Representative examples of transmission electron microscopic bright field imaging
of a partially-mineralized cusp region of tooth from row 16. (i), a longitudinal section of the
leading edge; (ii), longitudinal sections of the trailing edge.