Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry.
Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.
Hardt
Markus
M
Department of Cell and Tissue Biology, School of Dentistry, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA.
Thomas
Lindsay R
LR
Dixon
Scott E
SE
Newport
George
G
Agabian
Nina
N
Prakobphol
Akraporn
A
Hall
Steven C
SC
Witkowska
H Ewa
HE
Fisher
Susan J
SJ
D000328:Adult; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D054808:Histatins; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010306:Parotid Gland / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014462:Ultrafiltration
C068075:HTN3 protein, human; D054808:Histatins; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D020543:Proteome; D012471:Salivary Proteins and Peptides