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Salivary Proteomics: A Research Example

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Title: Salivary Proteomics: A Research Example


1
Salivary ProteomicsA Research Example
  • DENT 5302
  • Topics in Dental Biochemistry
  • Dr. Joel Rudney

2
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

3
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

4
http//chemfacilities.chem.indiana.edu/facilities/
proteomics/PRDFho1.gif
5
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

6
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

7
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

8
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

9
Results from resting whole saliva
10
Group differences - caries
11
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

12
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)

13
Biofilm DNA results
14
Results for total streptococci
15
T. forsythia results
16
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

17
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
18
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

19
Example from the basic pool
20
Reduced bands with VIP gt 0.80
21
Group differences for MAR9 and MAR10
22
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
23
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

24
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.
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