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

MOLAR OCCLUSAL SURFACES #DF 9 N = 37 7 N = 40 Max 75% Median 25% Min

TOP 25%

BOTTOM 25%

GROUPED BY ADHERENCE OF DEAD BACTERIA

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

7 6.7 6.5 6 5.7 5.3 4.7 4 3.5 3 NEUTRAL POOL MOD. ACIDIC ACIDIC POOL 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 Caries 0.63 0.18 Tf 0.82 0.26 Plaque 0.56 0.54 Strep 0.56 0.99 Group 0.54 0.50 Mean 0.62 0.49

B2
B16 MAR2 MAR3

0.89
0.51 0.71 0.81

1.00
0.53 0.58 0.87

0.93
0.44 0.52 0.59

0.81
0.31 0.55 0.59

0.47
0.96 0.80 0.57

0.82
0.55 0.63 0.67

MAR5
MAR6 MAR7 MAR9 NR2 NR3

0.45
0.71 0.85 0.73 0.27 0.80

0.44
0.58 0.59 0.90 0.99 0.65 0.86

0.61
0.52 0.43 0.92 0.70 0.54 0.57

1.01
0.55 0.40 0.91 0.65 0.55 1.07

0.31
0.80 0.84 0.73 1.03 0.82 0.26

0.56
0.63 0.62 0.84 0.84 0.57 0.71

MAR10 0.83

NR12

0.52

1.02

0.98

0.50

1.41

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.