4 Salivary Proteomics

8/12/2019 4 Salivary Proteomics

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Salivary Proteomics:

A Research Example

DENT 5302

Topics in Dental BiochemistryDr. Joel Rudney


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What is proteomics?

The goals of proteomics

Identify and catalog every protein in a biological system

Organs, diseases, cells, bacteria, biological fluids,

etc.Includes peptides, fragments, alleles, complexes

Compare proteome patterns

Cancer cells vs. control cells

Virulent 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


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Key proteomics technologies

Separating proteins along two dimensions

1-D separation - bands based on molecular weight

Different 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


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


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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 deadadherence

Define subjects at opposite extremes of function

Recall extreme subjects

Compare oral disease prevalence

Compare oral flora

Compare proteomic patterns


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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 analogThree 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


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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 swallowingDetermine 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


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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 species

Extract major components of common variation

A technique for simplifying complex data


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Results from resting whole saliva


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Group differences - caries

MOLAR OCCLUSAL SURFACES #DF

GROUPED BY ADHERENCE OF DEAD BACTERIA

BOTTOM 25%TOP 25%

9

7

5

3

1

N = 37 N = 40


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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


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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)


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Biofilm DNA results


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Results for total streptococci


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T. forsythiaresults


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Proteomic comparison

Recall 18 Haa and 23 Laa subjects

Collect fresh expectorated whole saliva

Clarify by centrifugation

Preparative isoelectric focusing - first dimensionBio-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


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20 fractions (from one subject)

11.5 10 9 8.7 8.48.2 8 7.7 7.4 7.2 7 6.7 6.5 6 5.7 5.34.7 4 3.5 3

BASICPOOL

NEUTRAL POOL MOD.ACIDIC

POOL

ACIDICPOOL


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Strategy for comparing subjects

For each pI pool

Molecular weight by SDS-PAGE - second dimension

Protein 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 AODBand matching by MW within and between group pairs

Partial least squares analysis

For when you have more variables than subjects


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Example from the basic pool


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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


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Group differences for MAR9 and MAR10

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


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Protein identification by MSMS

MAR9 is a truncated form of salivary cystatin S,missing the first 8 N-terminal amino acidsMAR10 is salivary statherin


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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 distributions

Associated with caries and microbiological outcomes

Markers for risk of caries and periodontal disease? Longitudinal studies needed

Clinically useful assays needed


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References

Rudney JD, Staikov RK (2002). Simultaneous measurement of the viability,

aggregation, and live and dead adherence of Streptococcus crista,

Streptococcus mutansandActinobacillus actinomycetemcomitansin

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 forsythensisand 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 salivaryantimicrobial functions. Presented at the 83rd General Session of theInternational Association for Dental Research, Baltimore, Maryland, March9-12, 2005.

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4 Salivary Proteomics