Supplementary Material
Spatial distribution of proteins in the quagga mussel adhesive apparatus
David J. Reesa, Arash Hanifia, Joseph Manionb, Arpita Gantayeta & Eli D. Sonea,b,c*
a
Institute of Biomaterials & Biomedical Engineering; University of Toronto, Toronto, ON, Canada
Department of Materials Science & Engineering, University of Toronto, Toronto, ON, Canada
c
Faculty of Dentistry; University of Toronto, Toronto, ON, Canada
b
1. Quagga mussel amino acid analysis quantification
To determine the effectiveness of our extraction procedure, we collected 15 induced thread/plaques (TP)
and performed amino acid analysis on the soluble protein component (supernatant, STP) and insoluble
protein component (protein pellet, ITP). Amino acid analysis was also performed on 10 TP homogenized
in 1% acetic acid with no further processing steps (whole thread/plaques, WTP). The results are
summarized below in Supplemental Table 1 and Supplemental Table 2. Notably, total DOPA content
is very low; the highest amount of DOPA observed was 0.14 mol% in the soluble protein component.
Table S1: Amino acid analysis performed on whole TP extract, soluble protein extract, and protein pellet.
Residue
ASX
GLX
SER
GLY
HIS
ARG
THR
ALA
PRO
DOPA
TYR
VAL
MET
CYS
ILE
LEU
PHE
LYS
Mol%
WTP
STP
ITP
12.4
8.7
5.4
15.5
1.0
3.0
5.3
4.6
6.2
0.01
7.2
8.3
1.9
0.6
6.1
7.1
3.2
3.5
10.5
6.3
5.5
25.3
2.7
4.5
3.0
5.4
9.3
0.11
7.3
4.0
0.8
1.2
2.5
5.0
2.1
4.6
6.7
4.0
3.5
16.1
1.7
2.8
1.9
3.4
6.6
0.14
6.4
12.4
2.6
0.8
10.0
10.7
4.7
5.4
Table S2: Overview of protein mass from quagga mussel induced whole TP, soluble extract, and insoluble.
Mass
WTP
STP
ITP
Normalized (μg/TP)
0.57
0.12
0.19
2. MALDI-TOF analysis using α-cyano matrix
Generally the spectra obtained using sinapinic acid matrix and α-cyano matrix were similar,
however α-cyano spectra had increased background signal noise. The α-cyano matrix did not
consistently ionize higher-weight proteins (>9kDa), however plaque specific peaks were
observed at ~13.5 kDa, ~15.8 kDa and ~18.0 kDa. A ~5.4 kDa peak had the highest signal
intensity in both the thread and plaque spectra, and thus may have a shared structure, shown
below in Supplemental Figure 1. Another high-intensity peak observed in plaque spectra was
centered at ~4.3 kDa: this peak also appeared in thread, upturned plaque, and plaque footprint
spectra, albeit with lower relative intensity (Supplemental Figure 2). The plaque-specific ~8.1
kDa protein appears to be present at the plaque interface, as it was observed in upturned plaque
and plaque footprint spectra. Furthermore, the ~6.5 - 6.7 kDa protein appears to be plaqueintensified, and also appears to be present at, or near, the plaque interface in upturned plaque
and plaque footprint spectra. As such, both the ~8.1 kDa and ~6.5 - 6.7 kDa proteins are
adhesive protein candidates. The ~5.4 kDa peak is also prominent at the interface, but given
that this peak also features prominently in thread spectra (Supplemental Fig. 2), it is less likely
that it plays a direct adhesive role.
2
Figure S1: MALDI-TOF representative spectra of isolated induced plaque and induced thread using α-cyano matrix. 6.1-6.7
kDa proteins appear with higher relative intensity in plaque. The ~5.4k Da and ~6.9 - 7.9 kDa proteins are shared between
plaque and thread spectra. Plaque-specific proteins are noted with red arrows: ~8.1, ~12-13.5, ~15.8, ~18.0 kDa.
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Figure S2: MALDI spectra from four sample preparations with α-cyano matrix. (A) Induced isolated plaque, (B) Upturned fresh
plaque, and (C) Fresh plaque footprint.
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3. Tricine PAGE separation of isolated threads and plaques:
In order to extract byssal proteins from threads and plaques separately, threads and plaques were
isolated from freshly secreted byssal material (using KCl induction), as described in the
Methods section. Proteins were extracted from isolated threads and plaques and separated using
Tricine PAGE, following the methods described in Gantayet et al. 2013 (Supplemental Figure
3) The lane containing isolated plaques produced similar bands the induced TP reported in the
paper body: Dbfp0 (>200 kDa), Dbfp1 (~69 kDa and ~80 kDa), Dbfp2 (~30 kDa), and Dbfp3
(~12 - 13 kDa, including the novel 6/7 kDa doublet, multiple bands from 14 - 20 kDa, and bands
at ~36 kDa and ~40 kDa. Notably, the lane containing isolated threads presented minimal
staining between the 6/7 kDa doublet and the intense ~36 kDa band. The Tricine PAGE results
support the MALDI-TOF observations that the proteins from ~8.1 – 14.8kDa are plaque-specific.
Figure S3: Tricine PAGE gel visualized with silver stain. Left lane contains protein standard ladder with mass
indicated in kDa, middle lane contains QM thread soluble protein, and right lane contains QM plaque soluble
proteins with labels indicating known/novel proteins.
Reference
Gantayet A, Ohana L, Sone ED. 2013. Byssal proteins of the freshwater zebra mussel, Dreissena polymorpha.
Biofouling 29:77-85.
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