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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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