Salivary Proteomics: A Research Example

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Salivary ProteomicsA Research Example

• DENT 5302
• Topics in Dental Biochemistry
• Dr. 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 strains
• Saliva 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 dead
  adherence
• 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 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

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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 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

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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 analysis
• Simultaneously 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
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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. forsythia results
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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
  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

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20 fractions (from one subject)
11.5
10
9
8.7
8.4
8.2
8
7.7
7.4
7.2
7
6.7
6.5
6
5.7
5.3
4.7
4
3.5
3
BASIC POOL
NEUTRAL POOL
MOD. ACIDIC POOL
ACIDIC POOL
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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 AOD
• Band 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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Reduced bands with VIP gt 0.80
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Group differences for MAR9 and MAR10
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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
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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 mutans and Actinobacillus
  actinomycetemcomitans in human saliva in relation
  to indices of caries, dental plaque and
  periodontal disease. Arch Oral Biol 47347-59.
• Rudney JD, Pan Y, Chen R (2003). Streptococcal
  diversity in oral biofilms with respect to
  salivary function. Arch Oral Biol 48475-93.
• 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
  49523-7.
• 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.