Salivary Proteomics:A Research Example

DENT 5302

Topics in Dental BiochemistryDr. Joel Rudney

What is proteomics? The goals of proteomics

Identify and catalog every protein in a biological systemOrgans, diseases, cells, bacteria, biological fluids, etc.Includes peptides, fragments, alleles, complexes

Compare proteome patternsCancer cells vs. control cellsVirulent bacteria vs. avirulent strainsSaliva from subjects w/ and w/o disease

• Biomarkers and diagnosis• Multifunctionality, amphifunctionality, redundancy

Salivary proteomics is a major research focus at NIDCR

Key proteomics technologies Separating proteins along two dimensions

1-D separation - bands based on molecular weightDifferent proteins with the same MW indistinguishable

2-D separation - MW vs IEP (charge)Much better resolution of different proteins (as spots)

Mass spectrometry Compare patterns, cut out and digest targets with trypsin Mass spectrometer gives exact MW of peptides in digest

Bioinformatics Derived protein sequences from human (& other) genomes Digest peptide pattern matched against all possibilities Precise identification usually possible

http://chemfacilities.chem.indiana.edu/facilities/proteomics/PRDFho1.gif

A research example Research problem - saliva proteins and oral health/ecology

Individual variation in individual salivary proteins Hard to relate to variation in oral flora and disease Multifunctionality, amphifunctionality, redundancy

Alternative strategy Measure individual variation in salivary functions

Bacterial killing, aggregation, live and dead adherence Define subjects at opposite “extremes” of function Recall “extreme” subjects

Compare oral disease prevalenceCompare oral floraCompare proteomic patterns

Measuring salivary function

Starting point: 96-well plate Coat the wells with hydroxyapatite

Add resting whole saliva - allow pellicle to form at 37° C Add equal volume of bacterial suspensions in saliva analog

Three different species used in different wells Streptococcus cristatus (commensal) Streptococcus mutans (caries) Actinobacillus actinomycetemcomitans (perio disease)

Add fluorescent live/dead DNA stains Blue “live” stain enters all bacteria If membrane damaged, green “dead” stain displaces “live”

Measurements of function

Aggregation Incubate in plate reader 4 hrs at 37° C Shake 1 sec every 2.5 min, read optical density

Shaking simulates shear force from swallowing Determine change in optical density over 4 hrs

Bacterial killing - read blue and green fluorescence Ratio of live to dead fluorescence after 4 hrs

Adherence of live and dead bacteria Wash plate - read blue and green fluorescence again

Adjust values for control wells Saliva only, bacteria only, buffer only

Study design Recruit two successive 1st-year dental classes

149 subjects consented Sample collection

Collect resting whole and stimulated parotid saliva Clinical exam for caries and periodontal indices

Assay saliva samples for three functions for each species Statistical analysis of the function data

Principal components analysisSimultaneously looks at variation in all variables

• 4 function variables x 3 speciesExtract major components of “common variation”A technique for simplifying complex data

Results from resting whole saliva

Group differences - caries

MOLAR OCCLUSAL SURFACES #DF

GROUPED BY ADHERENCE OF DEAD BACTERIA

Min25%Median75%Max

BOTTOM 25%TOP 25%

9

7

5

3

1

N = 37 N = 40

The recall phase

Recall students in the four extreme groups Collect resting whole saliva for proteomic study Collect overnight supragingival plaque for microbiology

Four sites exposed to different salivary flow• Buccal first molar site pooled• Lingual first molar sites pooled• Buccal upper incisor sites pooled• Lingual lower incisor sites pooled

Microbiology outcomes

Total biofilm DNA (proxy for total bacteria) Total streptococci (by quantitative PCR) Major periodontal pathogens (by quantitative PCR)

A. actinomycetemcomitans Porphyromonas gingivalis Tannerella forsythia (forsythensis)

Biofilm DNA results

Results for total streptococci

T. forsythia results

Proteomic comparison

Recall 18 Haa and 23 Laa subjects Collect fresh expectorated whole saliva

Clarify by centrifugation Preparative isoelectric focusing - first dimension

Bio-Rad Rotafor™ unit 20 fractions of different pI for each sample

Molecular weight by SDS-PAGE - second dimension Protein concentrations not standardized to preserve

quantitative differences

20 fractions (from one subject)

11.5 10 9 8.78.48.2 8 7.77.47.2 7 6.76.5 6 5.75.34.7 4 3.5 3

BASICPOOL

NEUTRAL POOL MOD.ACIDICPOOL

ACIDICPOOL

Strategy for comparing subjects For each pI pool

Molecular weight by SDS-PAGE - second dimensionProtein concentrations not standardized to preserve

quantitative differences Each sample replicated in three different gels

Gels for each group pair imaged Software used to determine:

Band MW and average optical density AOD Band matching by MW within and between group pairs

Partial least squares analysis For when you have more variables than subjects

Example from the basic pool

Band Caries Tf Plaque Strep Group Mean

AR4 0.63 0.82 0.56 0.56 0.54 0.62

B1 0.18 0.26 0.54 0.99 0.50 0.49

B2 0.89 1.00 0.93 0.81 0.47 0.82

B16 0.51 0.53 0.44 0.31 0.96 0.55

MAR2 0.71 0.58 0.52 0.55 0.80 0.63

MAR3 0.81 0.87 0.59 0.59 0.57 0.67

MAR5 0.45 0.44 0.61 1.01 0.31 0.56

MAR6 0.71 0.58 0.52 0.55 0.80 0.63

MAR7 0.85 0.59 0.43 0.40 0.84 0.62

MAR9 0.73 0.90 0.92 0.91 0.73 0.84

MAR10 0.83 0.99 0.70 0.65 1.03 0.84

NR2 0.27 0.65 0.54 0.55 0.82 0.57

NR3 0.80 0.86 0.57 1.07 0.26 0.71

NR12 0.52 1.02 0.98 0.50 1.41 0.89

Reduced bands with VIP > 0.80

Group differences for MAR9 and MAR10

t = –3.2; p = 0.0026 t = –5.7; p = 0.000001

Protein identification by MSMS

MAR9 is a truncated form of salivary cystatin S, missing the first 8 N-terminal amino acids

MAR10 is salivary statherin

Direct or indirect relationships?

Premature to assume direct relationships Intact cystatin S and statherin are pellicle components

Does variation in their prevalence affect pellicle structure? Could that in turn affect bacterial colonization patterns?

Direct relationships not essential to their use as biomarkers Desirable properties of N-8 cystatin S, and statherin

Broad continuous distributionsAssociated with caries and microbiological outcomes

• Markers for risk of caries and periodontal disease? Longitudinal studies needed Clinically useful assays needed

References

Rudney JD, Staikov RK (2002). Simultaneous measurement of the viability, aggregation, and live and dead adherence of Streptococcus crista, Streptococcus mutans and Actinobacillus actinomycetemcomitans in human saliva in relation to indices of caries, dental plaque and periodontal disease. Arch Oral Biol 47:347-59.

Rudney JD, Pan Y, Chen R (2003). Streptococcal diversity in oral biofilms with respect to salivary function. Arch Oral Biol 48:475-93.

Rudney JD, Chen R (2004). Human salivary function in relation to the prevalence of Tannerella forsythensis and other periodontal pathogens in early supragingival biofilm. Arch Oral Biol 49:523-7.

Rudney, J.D., R. K. Staikov, & Johnson, J.D. Proteomic analysis of salivary antimicrobial functions. Presented at the 83rd General Session of the International Association for Dental Research, Baltimore, Maryland, March 9-12, 2005.