Title: Salivary Proteomics: A Research Example
1Salivary ProteomicsA Research Example
- DENT 5302
- Topics in Dental Biochemistry
- Dr. Joel Rudney
2What 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
3Key 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
4http//chemfacilities.chem.indiana.edu/facilities/
proteomics/PRDFho1.gif
5A 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
6Measuring 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
7Measurements 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
8Study 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
9Results from resting whole saliva
10Group differences - caries
11The 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
12Microbiology 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)
13Biofilm DNA results
14Results for total streptococci
15T. forsythia results
16Proteomic 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
1720 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
18Strategy 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
19Example from the basic pool
20Reduced bands with VIP gt 0.80
21Group differences for MAR9 and MAR10
22Protein identification by MSMS
MAR9 is a truncated form of salivary cystatin S,
missing the first 8 N-terminal amino acids
MAR10 is salivary statherin
23Direct 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
24References
- 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.