Age-Dependent Modifications of the Human Salivary Secretory Protein Complex Tiziana Cabras,§ Elisabetta Pisano,† Roberto Boi,‡ Alessandra Olianas,§ Barbara Manconi,§ Rosanna Inzitari,‡,⊥ Chiara Fanali,‡,⊥ Bruno Giardina,‡,⊥,| Massimo Castagnola,‡,⊥,| and Irene Messana*,§ Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Universita` di Cagliari, Cagliari, Italy, Dipartimento di Chirurgia e Scienze Odontostomatologiche, Universita` di Cagliari, Cagliari, Italy, Istituto di Biochimica e Biochimica Clinica, Facolta` di Medicina, Universita` Cattolica, Rome, Italy, Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and Istituto Scientifico Internazionale “Paolo VI”, Rome, Italy Received March 4, 2009
Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/ peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups. Keywords: human saliva • age • salivary proline-rich proteins • histatins • proline-rich peptide P-B • statherin • salivary cystatins
Introduction The multifunctional nature of saliva is related to its heterogeneous composition, consisting of a complex mixture of proteins, peptides (comprised of enzymes and hormones), sugars, lipids, metal and nonmetal ions, and other components.1 The salivary proteome consists of components specifically secreted by major and minor salivary glands and components originating from other sources, such as the gingival crevicular fluid, desquamated epithelial cells, plasma exudation, and host * To whom correspondence should be addressed. Phone: +39-0706754520. Fax: +39-070-6754523. E-mail:
[email protected]. § Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Universita` di Cagliari. † Dipartimento di Chirurgia e Scienze Odontostomatologiche, Universita` di Cagliari. ‡ Istituto di Biochimica e Biochimica Clinica, Facolta` di Medicina, Universita` Cattolica. ⊥ Istituto per la Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche. | Istituto Scientifico Internazionale “Paolo VI”.
4126 Journal of Proteome Research 2009, 8, 4126–4134 Published on Web 06/25/2009
oral flora.2 Several proteomic studies evidenced this complexity, disclosing the presence in human saliva of more than 1400 different peptides and proteins.3-7 The major components secreted by the salivary glands and distinctive of human saliva can be grouped into a few families: histatins (Hst), statherin, proline-rich peptide P-B (P-B), S-type cystatins, acidic prolinerich phosphoproteins (aPRPs), basic salivary proline-rich proteins (bPRPs), basic glycosylated proline-rich proteins, amylases, and mucins.2 These salivary protein families are characterized by elevated genetic polymorphism as well as several pre- and postsecretory post-translational modifications, such as glycosylation, phosphorylation, transglutamination, sulfation, and proteolytic cleavages.2,5 Controversial findings are reported in the literature regarding the relationship between the age and the flow rate and composition of saliva in adults and children. In adults, secretion rates of unstimulated whole saliva were found to be negatively correlated with age both in males and females8,9 as well as females alone.10 Conversely, no significant influence of age on the stimulated salivary flow rate was observed in a cross10.1021/pr900212u CCC: $40.75
2009 American Chemical Society
Age-Related Trends of Human Salivary Proteins sectional study of unmedicated adults conducted by Parvinen and Larmas.11 Different results in adults were also obtained on the relationship between age and the concentration of either specific salivary proteins or total proteins in the whole saliva,12 parotid saliva,13-16 and submandibular/sublingual saliva.9,16 With regard to children, it was demonstrated that the flow rate increased in 10-year-old subjects during a 5-year followup. Further, the flow rate correlated negatively with protein levels.17 Similar results were obtained by Wu and co-workers,18 who reported an age-related flow rate increase and protein level decrease in the unstimulated saliva of preschool and elementary school groups.18 Conversely, Rotteveel et al. observed that age did not influence the salivary flow rate in healthy children aged 6-11 years.19 Salivary protein concentrations rose linearly with age, but no differences were found between age groups in a study performed on 136 individuals aged from 7 months to 63 years.20 Finally, our group demonstrated that the salivary protein profiles of newborns and adults show remarkable differences.21 In the present study, we proposed to investigate the qualitative and quantitative changes occurring in the secretion of proteins and peptides specific to the oral cavity soluble in acidic solution (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) with respect to age from the pediatric to the postpuberty period. To this end, the acid-soluble fraction of saliva collected from a group of healthy subjects aged from 3 to 44 years was analyzed by RP-HPLC-ESI-MS. This method has been proven to be a suitable tool for the study of naturally occurring proteins and peptides in saliva.22-25 A contribution to the knowledge regarding physiological variability in the human salivary proteome is important for reaching the goal of using saliva as a diagnostic and prognostic fluid.
Materials and Methods Reagents and Instruments. All analytical grade common chemicals and reagents were purchased from Farmitalia-Carlo Erba (Milan, Italy), Merck (Darmstadt, Germany), and Sigma Aldrich (St. Louis, MI, USA). The HPLC-ESI-IT-MS apparatus is a Surveyor HPLC system (ThermoFisher, San Jose, CA, USA) connected by a T splitter to a photo diode-array detector and to a LCQ Deca XP Plus mass spectrometer. The MS apparatus was equipped with an electrospray ionization (ESI) source. The chromatographic column was a 150 × 2.1 mm Vydac (Hesperia, CA, USA) C8 column, with 5-µm particle diameter. Sample Collection and Treatment. The study protocol and written consent form were approved by the Paediatric Department Ethics Committee and by the Medical Committee of the Faculty of Medicine of the Catholic University of Rome (according to the instructions of the Declaration of Helsinki). All rules were respected, and written consent forms were signed by either volunteers or parents of children. Unstimulated whole saliva (WS) was collected from 67 informed donors (age 3-44 years) divided according to age into five groups: group A, 8 donors, 3 females (F) and 5 males (M), 3.6 ( 0.9 year (mean age ( SD); group B, 11 donors, 6 F and 5 M, 7.5 ( 1.2 years; group C, 20 donors, 9 F and 11 M, 10.9 ( 0.8 years; group D, 15 donors, 7 F and 8 M, 14.9 ( 1.5 years; group E, 13 donors, 7 F and 6 M, 34.5 ( 9.7 years. Collection time was established between 10.00 and 12.00 a.m.. The donors were in healthy
research articles clinical conditions, not affected by pathologies of either the salivary glands or the oral cavity; adult donors were nonsmokers. Donors did not eat or drink 2 h before the collection. The donors were invited to sit, assuming a relaxed position, and to swallow. Then, WS was collected with a soft plastic aspirator as it flowed into the anterior floor of the mouth. Samples were not pooled, but they were treated and analyzed separately. After collection, salivary samples were immediately diluted in a 1:1 v/v ratio with 0.2% aqueous TFA in an ice bath. The solution was centrifuged at 8000 g for 5 min (4 °C). The acidic supernatant was separated from the precipitate and either immediately analyzed by the HPLC-ESI-MS apparatus (100 µL, corresponding to 50 µL of saliva) or stored at -80 °C until the analysis. RP-HPLC-ESI-MS Analysis. The following solutions were utilized for the RP-HPLC separation: (eluent A) 0.056% aqueous TFA and (eluent B) 0.05% TFA in acetonitrile-water 80/20. The proteins were eluted by using a linear gradient from 0% to 55% of B for 40 min, at a flow rate of 0.30 mL/min. The T splitter permitted 0.20 mL/min to flow toward the diode array detector and 0.10 mL/min to flow toward the ESI source. The diode array detector was set at 214 and 276 nm. Mass spectra were collected every 3 ms in the positive ion mode. The MS spray voltage was 4.50 kV, and the capillary temperature was 220 °C. The structure of new peptides was confirmed by Tandem/ MS experiments, performed on doubly and triply charged ions. Detection utilized a peak width of 2-4 m/z value and 40% of the maximum activation amplitude. Some samples were also analyzed by an Ultimate 3000 Nano/ Micro HPLC apparatus (Dionex, Sunnyvale, CA, USA) equipped with a FLM-3000-Flow manager module coupled to a LTQ Orbitrap XL apparatus (ThermoFisher, San Jose, CA, USA). The column was a Biobasic 8 capillary column with a 3 µm particle diameter (column dimension 180 µm i.d. × 10 cm). The chromatographic eluents were as follows: (A) 0.1% aqueous formic acid and (B) 0.1% formic acid in acetonitrile. The applied gradient was 0-4 min 5% B, 4-38 min from 5% to 50% B (linear), and 38-41 min from 50% to 90% B (linear) at a flow rate of 4 µL/min. Mass spectra were collected in full scan mode (MS data) and also in data-dependent scan mode (MS/MS data) with a capillary temperature of 250 °C, a sheath gas flow of 17 arbitrary units, a source voltage of 3.6 kV, and a capillary voltage of 32 V. The mass accuracy (FT) was calibrated before measurements that were performed in the positive ion mode. Selected protein charge states were isolated with a width of m/z 6-10 and activated for 30 ms, using 30% normalized collision energy and an activation q of 0.25. Quantification. The total protein concentration of the acidsoluble fraction was determined in duplicate by the bicinchoninic acid assay (Micro BCA Protein Assay kit by PierceThermoFisher Scientific, Rockford, IL, USA) according to manufacturer’s instructions. Quantification of specific proteins and peptides was based on the area of the RP-HPLC-ESI-MS eXtracted Ion Current (XIC) peaks. XIC analysis selectively reveals a protein in the chromatographic profile by extracting the ion current associated with the multiply charged ions characteristic of the protein. The ions utilized to reveal the proteins/peptides were carefully selected in order to exclude values in common with other coeluting proteins (Table 1). Under constant analytical conditions, the area of the extracted ion current peaks is proportional to the peptide/protein concentration.2 Journal of Proteome Research • Vol. 8, No. 8, 2009 4127
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Table 1. Average Mass Values (Experimental and Theoretical), Elution Times, and Multiply-Charged Ions Used to Reveal XIC Peaks of the Proteins and Peptides Quantified protein (Swiss-Prot code or ref)
elution time (min)
exptl av mass (theor av mass)
m/z values of multiply charged ions used to extract ion current peaks (charge in parentheses)
basic salivary proline-rich proteins 5843.0 ( 0.5 (5842.5) 1461.6 (+4), 1169.5 (+5), 974.8 (+6), 835.6 (+7), 731.3 (+8). 5943.9 ( 0.5 (5943.6) 1486.9 (+4), 1189.7 (+5), 991.6 (+6), 850.1 (+7), 743.9 (+8). 5590.2 ( 0.5 (5590.1) 1398.5 (+4), 1119.0 (+5), 932.7 (+6), 799.6 (+7), 699.8 (+8). 11898 ( 2 (11896.2) 1082.5 (+11), 992.4 (+12), 916.1 (+13), 850.7 (+14), 794.1 (+15), 744.5 (+16). 5769.0 ( 0.5 (5769.4) 1443.4 (+4), 1154.9 (+5), 962.6 (+6), 825.2 (+7), 722.2 (+8). 9593 ( 1 (9593.4) 1919.7 (+5), 1599.9 (+6), 1371.5 (+7), 1200.2 (+8), 960.3 (+10), 873.1 (+11). 9513 ( 1 (9513.4) 1586.6 (+6), 1360.1 (+7), 1190.2 (+8), 1058.0 (+9), 865.9 (+11). 9437 ( 1 (9437.2) 1573.9 (+6), 1349.2 (+7), 1180.7 (+8), 1049.6 (+9), 944.7 (+10). 11517 ( 2 (11517) 1152.7 (+10), 1048.0 (+11), 886.9 (+13), 823.6 (+14), 768.8 (+16). 7609 ( 1 (7609.2) 1903.3 (+4), 1522.8 (+5), 1269.2 (+6), 1088.0 (+7), 952.2 (+8), 846.5 (+9). 7529 ( 1 (7529.2) 1883.3 (+4), 1506.8 (+5), 1255.9 (+6), 1076.6 (+7), 942.1 (+8). 7453 ( 1 (7453.0) 1864.3 (+4), 1491.6 (+5), 1243.2 (+6), 1065.7 (+7), 932.6 (+8). 6024 ( 1 (6023.7) 1507.0 (+4), 1205.8 (+5), 1005.0 (+6), 861.6 (+7), 754.0 (+8). 6950 ( 1 (6949.7) 1738.4 (+4), 1390.9 (+5), 1159.3 (+6), 993.8 (+7), 869.7 (+8), 773.2 (+9).
P-F (IB8-c) (P02812) P-Ja P-H (IB-4) (P02812/P04280)b IB8-a (Con1-)c
14.3-14.8 14.1-14.7 15.0-15.5 17.1-17.8
IB7 (P02812) IB-1 (P04281)
14.2-14.6 18.8-19.3
IB-1 nonphosd IB-1 Des-Arg96d IB-6 (P04280) II-2 (P04280)
19.4-19.7 19.0-19.4 16.3-16.9 18.7-19.1
II-2 nonphosd II-2 Des-Arg75d P-E (IB-9) (P02811) P-D (IB-5) (P10163)
19.5-19.8 18.8-19.2 14.7-15.3 15.2-15.8
PRP-1 type diphos (P02810) PRP-1 type monophos PRP-1 type nonphos PRP-1 type triphos PRP-3 type diphos (P02810) PRP-3 type monophos PRP-3 type nonphos PRP-3 type diphos Des-Arg106 P-C peptide (P02810) Db-s diphos
22.9-23.3 23.9-24.3 24.2-24.7 22.6-22.9 23.3-23.8 23.8-24.2 24.8-25.1 23.5-23.8 13.6-14.5 22.9-23.2
Db-s monophos
23.4-23.8
Db-s tri-phos
22.6-23.1
Db-f diphos
23.1-23.5
Db-f monophos
23.6-24.1
Pa 2-mer
24.6-25.1
Hst-1 Hst-1 Hst-3 Hst-3 Hst-3
23.3-23.8 23.4-23.8 17.6-17.9 14.0-14.4 14.2-14.7
histatin family 4928.2 ( 0.5 (4928.2) 1644.1 4848.2 ( 0.5 (4848.2) 1617.4 4062.2 ( 0.4 (4062.4) 1355.1 3192.4 ( 0.3 (3192.5) 1065.1 3036.5 ( 0.3 (3036.3) 1013.2
29.4-30.5 29.9-30.7 29.6-30.5 29.6-30.2 27.2-27.7
proline-rich peptide P-B family 5792.9 ( 0.5 (5792.7) 1932.0 (+3), 1449.2 5215.0 ( 0.5 (5215.1) 1739.4 (+3), 1304.8 5062.0 ( 0.5 (5060.9) 1688.0 (+3), 1266.2 5370.7 ( 0.5 (5371.3) 1791.2 (+3), 1343.7 4549.4 ( 0.5 (4549.3) 1517.3 (+3), 1138.3
28.9-29.5 28.7-29.1 28.4-28.8 27.6-28.0 27.7-28.1 28.5-28.9 27.5-28.8 27.8-28.2 27.8-28.3
statherin family 5380.0 ( 0.5 (5379.7) 1794.2 5299.9 ( 0.5 (5299.7) 1767.6 5220.5 ( 0.5 (5219.7) 1741.2 5232.4 ( 0.5 (5232.5) 1745.1 5131.2 ( 0.5 (5131.4) 1711.4 5264.7 ( 0.5 (5264.6) 1755.9 4127.9 ( 0.4 (4127.6) 1376.9 3971.3 ( 0.4 (3971.4) 1986.7 3645.2 ( 0.4 (3645.0) 1823.6
P-B P-B P-B P-B P-B
(P15515) nonphos (P15515) (P15516) 1/25 (P15516) 1/24 (P15516)
(P02814) Des1-5f Des1-7 Des1-4 Des1-12
statherin diphos (P02808) statherin monophosf statherin nonphosf SV1 (Des-Phe43)g statherin Des-Thr42Phe43f statherin Des-Asp1h statherin Des1-9f Statherin Des1-10f statherin Des1-13f
salivary acidic proline-rich phosphoproteinse 15515 ( 2 (15514-15515) 1293.9 (+12), 1194.4 (+13), 1035.3 (+15), 970.7 (+16), 913.6 (+17). 15435 ( 2 ( 15434-15435) 1287.2 (+12), 1188.3 (+13), 1030.0 (+15), 965.7 (+16), 908.9 (+17). 15355 ( 2 (15354-15355) 1280.5 (+12), 1182.1 (+13), 1024.6 (+15), 960.7 (+16), 904.2 (+17). 15595 ( 2 ( 15594-15595) 1418.7 (+11), 1300.5 (+12), 1200.6 (+13), 1040.6 (+15), 975.7 (+16). 11161 ( 1 (11161-11162) 1595.5 (+7), 1396.2 (+8), 1015.7 (+11), 931.1 (+12), 859.6 (+13). 11081 ( 1 ( 11081-11082) 1584.1 (+7), 1386.2 (+8), 1008.4 (+11), 924.5 (+12), 853.4 (+13). 11001 ( 1 (11001-11002) 1376.2 (+8), 1101.2 (+10), 917.8 (+12) 786.8 (+14). 11004 ( 1 (11005-11006) 1573.2 (+7), 1223.8 (+9), 1001.5 (+11), 847.6 (+13). 4370.9 ( 0.4 (4370.8) 1457.9 (+3), 1093.7 (+4). 17633 ( 2 (17633) 1604.0 (+11), 1470.4 (+12), 1357.4 (+13), 1260.5 (+14), 1176.5 (+15), 1038.2 (+17). 17553 ( 2 (17533) 1756.3 (+10), 1463.8 (+12), 1351.2 (+13), 1254.8 (+14), 1171.2 (+15), 1098.2 (+16). 17713 ( 2 (17713) 1772.3 (+10), 1611.3 (+11), 1477.1 (+12), 1363.5 (+13), 1266.2 (+14), 1042.9 (+17). 13280 ( 2 (13280) 1661.0 (+8), 1329.0 (+10), 1208.3 (+11), 1107.7 (+12), 1022.5 (+13), 949.6 (+14). 13200 ( 2 (13200) 1886.7 (+7), 1651.0 (+8), 1467.7 (+9), 1321.0 (+10), 943.9 (+14), 881.0 (+15). 30922 ( 3 (30922) 1628.5 (+19), 1547.1 (+20), 1473.5 (+21), 1406.6 (+22), 1345.4 (+23), 1237.9 (+25), 1146.3 (+27). (+3), (+3), (+3), (+3), (+3),
(+3), (+3), (+3), (+3), (+3), (+3), (+3), (+2), (+2),
1233.5 (+4). 1213.5 (+4). 1016.6 (+4). 799.1 (+4). 760.1 (+4). (+4), (+4), (+4), (+4), (+4).
1159.6 1044.0 1013.2 1075.1
1345.9 (+4), 1076.9 (+5). 1325.9(+4), 1060.9 (+5). 1306.1 (+4), 1045.1 (+5). 1309.1(+4), 1047.5 (+5). 1283.8 (+4), 1027.2 (+5). 1317.2 (+4), 1053.9 (+5). 1032.9 (+4). 1324.8 (+3). 1216.1 (+3).
salivary cystatin family 36.5-37.1 14186 ( 2 (14185) 1774.3 (+8), 1577.2 (+9), 1419.6 1092.2 (+13), 1014.3 (+14). S monophos at Ser3 (P01036) 36.6-37.1 14266 ( 2 (14265) 1784.3 (+8), 1586.1 (+9), 1427.6 1098.4 (+13), 1020.0 (+14). S diphos at Ser1 and Ser3 (P01036) 36.8-37.2 14346 ( 2 (14345) 1794.3 (+8), 1595.0 (+9), 1435.6 1104.5 (+13), 1025.7 (+14). SN (P01037) 34.8-35.2 14312 ( 2 (14313) 1790.0 (+8), 1591.2 (+9), 1432.2 1101.9 (+13), 1023.3 (+14). SA (P09228) 38.4-38.9 14347 ( 2 (14346) 1794.4 (+8), 1595.1 (+9), 1435.7 1104.6 (+13), 1025.8 (+14). S nonphos (P01036)
(+5). (+5). (+5). (+5).
(+10), 1290.6 (+11), 1183.2 (+12), (+10), 1297.9 (+11), 1189.8 (+12), (+10), 1305.2 (+11), 1196.5 (+12), (+10), 1302.1 (+11), 1193.7 (+12), (+10), 1305.3 (+11), 1196.6 (+12),
a Characterized in the present work. b Reference 32. c Reference 29. d Reference 22. e The term PRP-1 type includes the three entire isoforms PRP-1 and PRP-2 (expressed by the PRH2 locus) as well as Pif-s (expressed by the PRH1 locus), with a mass difference of 1 Da. The term PRP-3 type includes the truncated isoforms PRP-3, PRP-4, and Pif-f (ref 24). f Reference 44. g Reference 45. h Reference 46.
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Age-Related Trends of Human Salivary Proteins
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Data Analysis and Statistics. Deconvolution of the averaged ESI-MS spectra was automatically performed by using either the Bioworks Browser software provided with the LCQ Deca XP instrument or MagTran 1.0 software.26 Experimental mass values were compared with average theoretical values available at the Swiss-Prot Data Bank (http://us.expasy.org/tools) with the codes reported in Table 1 using the PeptideMass and FindPept programs. Peptides not previously identified were characterized by manual comparison of their experimental MS/ MS spectra with the theoretical spectra generated by using the MS-Product program available with ProteinProspector. Deconvolution of Orbitrap MS/MS data was performed with the software provided with the LTQ Orbitrap XL (Xtract on QualBrowser 2.0). The software GraphPad Prism (version 4.0) was used for statistical analysis. The nonparametric Mann-Whitney test was used to compare the five age groups. Statistical analysis was considered significant when the p-value was
