We thank the Office of Naval Research and the NREIP program for financial support. We thank Lenora Brewer (Cal Poly), Eric Holm (NSWC Carderock), Gary Dickenson, Beatriz Orihuela, and Dan Rittschof (all from Duke University), and the entire DEW lab.

Proteomic Approach for Investigating Temperature Effects on Barnacle (Balanus amphitrite) Cement Proteins

Daugherty, Melissa J., Laurel Johnston, Lenora Brewer, and Dean E. WendtCenter for Coastal Marine Sciences

California Polytechnic State University, San Luis Obispo, USA

Acknowledgments

Initial Analysis: Cement Verification1D SDS PAGE Analysis of Cement, Body Tissues, and Fluids

Richard Fox, 2001 Lander University

Body Fluid (hemolymph) CollectionUncured Cement Collection Dissection & Tissue Collection

Expected Outcomes• Verification of organismal regulation of composition or quality of cement/adhesive

• Detection of changes in cement protein expression as a function of temperature

• Detection of post translational modifications in known proteins.

• Possible identification of novel cement proteins not previously characterized

• Increased understanding of mechanism of attachment to improve coating design

Experimental Design: Assessment of Rearing Temperature on Expression of Barnacle

Cement Proteins

Microslides Silastic® T2 (Dow Corning)

25oC

Put in Petri

dishes

Incubate for 72 hours

Distribute slides

between Temperature

s

Cyprid larvae

Fed Skeletonema costatum and Dunaliella

tertiolecta

15oC 25oCFed Artemia sp.

after 1 month

Collect cement from individuals of each

treatment

2D Gel Electrophoresis25°C

2D Gel Electrophoresis15°C

Gel Comparison and

AnalysisFig 3. Experimental design flowchart.

Protein Sample Prep Protein Separation

PEP Analysis

MS Analysis

+

-

1st Dimension pH Gradient pH 3-102nd

DimensionMolecular Weight Gradient

Proteomic Analysis

Spot Cutting & Digest

Data Processing

Laser

Mass AnalyzerMatrix

Sample Ions

Ion source

Ionization(charged molecules)

Ion Detector

Detection

Mass Analyzer

Mass sorting

1530

.686

1456

.746

1784

.901

2839

.284

984.

489

1323

.649

2211

.104

679.

504

0.00

0.25

0.50

0.75

1.00

1.25

1.50

4x10

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500 4000 4500m/z

PMFPeptides and their masses

1530

.686

1456

.746

1784

.901

2839

.284

984.

489

1323

.649

1560

.696

2211

.104

679.

50451

5.06

9

928.

895

1483

.679

F12\0_F12\1\1SRef

0.0

0.5

1.0

1.5

4x10

Inte

ns. [

a.u.

]

175.

196

359.

325

531.

408

460.

378

288.

285

1438

.532

86.1

59

234.

199

F12 LIFT 1530\0_F12\1\1530.6860.LIFT\1SRef

0

200

400

600

Inte

ns. [

a.u.

]

500 1000 1500 2000 2500m/z

Select peptide for tandem MS/MS Further

dissociation

De novosequencing Scores associated

with probability

Database search based

on peptide/sequence

similarity

Fig 5. Proteomic analysis flowchart.

DECODON Delta2D

Literature CitedKamino K. 2008. Underwater adhesive of marine organisms as the vital link between biological science and material science. Mar. Biotechnol. 10:111-121. Kamino K. 2006. Barnacle underwater attachment. In: Smith AM, Callow JA (eds) Biological adhesives. Springer- Cerlag, Berlin. pp 145-166. Kamino, K. 2001. Novel barnacle underwater adhesive protein is a charged amino acid-rich protein

constituted by a Cys-rich repetitive sequence. Biochem. J. 356:503-507.Kamino, K., K. Inoue, T. Maruyama, N. Takamatsu, S. Harayama, and Y. Shizuri. 2000. Barnacle cement

proteins. J. Biol. Chem. 275:27360–27365.Kamino, K., S. Odo, and T. Maruyama. 1996. Cement proteins of the acorn barnacle, Megabalanus

rosa. Biol. Bull. 190:403-409.Khandeparker, L., and A. C. Anil. 2007. Underwater adhesion: the barnacle way. Int. J. Adhesion. Adhes. 27:165- 172.Naldrett, M. J., and D. L. Kaplan. 1997. Characterization of barnacle (Balanus eburneus and B. cenatus) adhesive proteins. Mar. Biol. 127:629-635.Urushida, Y., M. Nakano, S. Matsuda, N. Inoue, S. Kanai, N. Kitamura, T. Nishino, and K. Kamino. 2007. Identification and functional characterization of a novel barnacle cement protein. FEBS. J. 274:4336-4346.

Fig 2. Flowchart of initial sample analysis: liquid cement will be collected by mechanical probing of basal disc, soft tissues will be dissected out and homogenized, liquid body fluid will be extracted from the mantle cavity using a hypodermic needle. All samples will be run out on 1D SDS PAGE gels for comparison of variation in protein content between samples in order to confirm cement collection technique.

Barnacles are often the dominant hard foulers in marine waters and they attach to substrates by secreting a proteinaceous adhesive (Kamino, 2001, 2006, 2008; Naldret and Kaplan, 1997; Kamino et al., 1996, 2000). Understanding the chemical composition of their underwater adhesive is central to developing non-toxic solutions to control biofouling (Khandeparker et al., 2007).

Approximately ten cement proteins (CPs) have been recognized. Of these, six have been identified and grouped into four different categories: hydrophobic proteins, six amino acid-biased proteins, a charged amino acid-rich protein, and an enzyme. Furthermore, five out of the six CPs (with the exception of the enzyme) are novel in their primary structure, confirming the uniqueness of this cement protein complex (see Kamino 2006 for review). Additional research investigating the molecular interactions between the constituents of this multi-protein adhesive will provide valuable insight for the development of more effective antifouling materials.

Environmental variables such as temperature and salinity may influence the adhesive properties of barnacle cement. Indeed, recent experiments in our lab have shown the critical removal stress (CRS) for barnacles reared at different temperatures demonstrated an inverse relationship between CRS and temperature (Fig 1). The mechanism accounting for this trend is still unknown and could be attributed to compositional changes in the adhesive as a result of temperature. Preliminary experiments using 1D SDS PAGE confirmed the presence of multiple proteins in uncured cement. Over the next two years we are extending the previous experiments to include a proteomic approach of 2D Gel Electrophoresis to explore potential variations in cement protein composition among animals grown at different temperatures.

The proposed study will focus on the collection of uncured cement and utilize proteomic analysis to identify and characterize the expression of cement proteins. Specifically, we expect to: 1) monitor changes in protein expression due to rearing temperatures 15C and 25C; 2) identify and infer functions of individual proteins in response to these varying temperatures based on similarity and comparisons of other known proteins; and, 3) document and catalog novel proteins using high-performance mass spectrometry and de novo sequencing; a process that further dissociates initial peptide fragments generating specific amino acid sequences, which can then be searched for sequence similarity of known protein origin. This proteomic approach will help elucidate the effects of environmental parameters on the adhesive abilities of this ubiquitous and problematic fouling species.

Average Critical Removal Stress at Three Temperatures

0

0.05

0.1

0.15

0.2

0.25

Fifteen Twenty-five Thirty

Temperature (°C)

CR

S (

N/m

m2 )

n=31 n=36 n=57

Fig 1. Average Critical removal stress showed an inverse relationship with temperature. Post-hoc analysis showed significant differences between all temperatures p= <0.0001 F-stat= 14.675

n=31 n=57n=36

Introduction

Proteomic Workflow

Identification of proteins

(with mass spectrometry)

Spot cutting (out of the gel)

Trypsin-digestion

Gel image analysis: spot detection, matching and warping

Quantification of protein expression profiles (PEPs)

Protein separation: SDS-PAGE (2nd dimension) + staining

Protein solubilization (pre-fractionation)

Protein separation: Isoelectric focusing (IEF)

(1st dimension)

Fig 4. Proteomic flowchart.