Salivary Proteomics: A Research Example DENT 5302 Topics in Dental Biochemistry Dr. Joel Rudney

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

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

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

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

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”

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

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

Results from resting whole saliva

Group differences - caries

3 1 9 MOLAR OCCLUSAL SURFACES #DF N = 37 N = 40 7 5 TOP 25% BOTTOM 25% GROUPED BY ADHERENCE OF DEAD BACTERIA Max 75% Median 25% Min

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

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)

Biofilm DNA results

Results for total streptococci

T. forsythia

results

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

20 fractions (from one subject)

11.5 10 9 8.7 8.4 8.2 8 7.7 7.4 7.2

BASIC POOL NEUTRAL POOL 7 6.7 6.5 6 5.7 5.3 4.7 4 3.5 3 MOD.

ACIDIC POOL ACIDIC POOL

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

Example from the basic pool

Reduced bands with VIP > 0.80

Band AR4 B1 B2 B16 MAR2 MAR3 MAR5 MAR6 MAR7 MAR9 MAR10 NR2 NR3 NR12 Caries 0.63

0.18

0.89

0.51

0.71

0.81

0.45

0.71

0.85

0.73

0.83

0.27

0.80

0.52

Tf 0.82

0.26

1.00

0.53

0.58

0.87

0.44

0.58

0.59

0.90

0.99

0.65

0.86

1.02

Plaque 0.56

0.54

0.93

0.44

0.52

0.59

0.61

0.52

0.43

0.92

0.70

0.54

0.57

0.98

Strep 0.56

0.99

0.81

0.31

0.55

0.59

1.01

0.55

0.40

0.91

0.65

0.55

1.07

0.50

Group 0.54

0.50

0.47

0.96

0.80

0.57

0.31

0.80

0.84

0.73

1.03

0.82

0.26

1.41

Mean 0.62

0.49

0.82

0.55

0.63

0.67

0.56

0.63

0.62

0.84

0.84

0.57

0.71

0.89

Group differences for MAR9 and MAR10

t = –3.2; p = 0.0026

t = –5.7; p = 0.000001

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

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

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 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 forsythensis

and 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 salivary antimicrobial functions. Presented at the 83rd General Session of the International Association for Dental Research, Baltimore, Maryland, March 9-12, 2005.