Quantitative Analysis of Age Specific Variation in the Abundance of Human Female Parotid Salivary Proteins

Center for Oral Biology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Journal of Proteome Research (Impact Factor: 4.25). 09/2009; 8(11):5093-102. DOI: 10.1021/pr900478h
Source: PubMed


Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however, little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20-30 and 55-65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high- from low-abundance proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g., lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by Western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women.

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    • "After the collection and pooling of human whole saliva, the protein concentrations and composition of pooled saliva from the six age and gender groups were measured by BCA protein assay and SDS-PAGE, respectively. The overall trend was that the mean protein concentration of human whole saliva increased with age (Figure 1), in agreement with the results of a previous report [33]. The increasing rate was higher in females than in males. "
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    ABSTRACT: Background Glycoproteins comprise a large portion of the salivary proteome and have great potential for biomarker discovery and disease diagnosis. However, the rate of production and the concentration of whole saliva change with age, gender and physiological states of the human body. Therefore, a thorough understanding of the salivary glycoproteome of healthy individuals of different ages and genders is a prerequisite for saliva to have clinical utility. Methods Formerly N-linked glycopeptides were isolated from the pooled whole saliva of six age and gender groups by hydrazide chemistry and hydrophilic affinity methods followed by mass spectrometry identification. Selected physiochemical characteristics of salivary glycoproteins were analyzed, and the salivary glycoproteomes of different age and gender groups were compared based on their glycoprotein components and gene ontology. Results and discussion Among 85 N-glycoproteins identified in healthy human saliva, the majority were acidic proteins with low molecular weight. The numbers of salivary N-glycoproteins increased with age. Fifteen salivary glycoproteins were identified as potential age- or gender-associated glycoproteins, and many of them have functions related to innate immunity against microorganisms and oral cavity protection. Moreover, many salivary glycoproteins have been previously reported as disease related glycoproteins. This study reveals the important role of salivary glycoproteins in the maintenance of oral health and homeostasis and the great potential of saliva for biomarker discovery and disease diagnosis.
    06/2014; 11(1-1):25. DOI:10.1186/1559-0275-11-25
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    • "All the saliva samples were collected between 9 am - 12 pm to minimise diurnal variations associated with saliva sampling. In addition, both females and male participants were <35 years of age to minimise age related differences in the salivary biomolecular composition [22,23]. The participants rinsed their mouth with water prior to collection, and waited 10 minutes before commencing with the collection. "
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    ABSTRACT: Background Owing to its ease of collection, saliva is potentially the sample of choice in diagnosis. Salivary biomolecules have provided a porthole in surveying a person’s health and well-being. Our study aims were (1) to demonstrate the effects of pre-analytical steps, collection and pre-processing techniques on salivary protein detection and (2) to establish an indication of salivary reference intervals for 3 biomolecules of clinical interest. Methods Saliva samples were collected from participants (n = 25, ages 20–35 years) using the following methods: no stimulation (resting/unstimulated), mechanical, and acid stimulation. The saliva was prepared for analysis by: unprocessed, post standard centrifugation in a container without any additives, and centrifugation using Centrifugal Filter Unit (Amicon® Ultra-0.5). AlphaLisa® assays were used to measure the levels of C-Reactive Protein (CRP), Immunoglobin (IgE) and myoglobin in saliva samples. Results Saliva flow rates were lowest with the resting/drooling collection method. The lowest total protein concentration was with acid stimulation. Unstimulated and mechanically stimulated collections produced no effect on the CRP and IgE levels while myoglobin levels were highest with the unstimulated collection. Acid stimulation had a negative impact on the measured concentrations of IgE and myoglobin (except for CRP levels). Conclusion Mechanical stimulation was the most viable option for collecting saliva without affecting the levels of CRP and myoglobin. The processing methods had an adverse effect on the concentration of total protein as well as on CRP and IgE concentrations.
    Clinical and Translational Medicine 09/2012; 1(1):19. DOI:10.1186/2001-1326-1-19
    • "Hydrophobic interaction chromatography, where adsorption occurs by the formation of water-repelling associations , is developed by using lyotropic decreasing gradients or hydro-organic mixed eluents. Changes of certain protein abundances have been discovered between young and old individuals by fractionating 14 samples of saliva proteins by hydrophobic charge induction chromatography prior to full digestion of collected fractions and multidimensional protein fractionation and identification technology [25]. More proteins were found in the aged individuals (460 versus 338 gene products with 266 in common) and differentially expressed proteins were found especially related to defense such as lysozyme, lactoferrin and histatin-1. "
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    ABSTRACT: The discovery phase of biomarkers of diagnostic or therapeutic interest started a decade ago with the very rapid development of proteomic investigations. In spite of the development of innovative technologies and multiple approaches, the "harvest" is still modest. Various reasons justified the encountered difficulties and most of them have been circumvented by specific sample treatments or dedicated analytical approaches. Nevertheless, the situation of very modest biomarker discovery level did not change much. This review intends to specifically analyze the main approaches used for biomarker discovery phase and evaluate related advantages and disadvantages. Thus, preliminary sample treatments such as fractionation, depletion and reduction of dynamic concentration range will critically be discussed and then the main differential expression investigation methods analyzed. Combinations of technologies are also discussed along with possible proposals to federate associations of complementary technologies for better chances of success.
    PROTEOMICS - CLINICAL APPLICATIONS 01/2012; 6(1-2):22-41. DOI:10.1002/prca.201100039 · 2.96 Impact Factor
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