Quantitative analysis of the intra- and inter-subject variability of the whole salivary proteome
N. Jehmlich
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorK. H. D. Dinh
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorM. Gesell-Salazar
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorE. Hammer
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorL. Steil
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorV. M. Dhople
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorC. Schurmann
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorB. Holtfreter
Unit of Periodontology, Department of Restorative Dentistry, Periodontology and Endodontology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany
Search for more papers by this authorT. Kocher
Unit of Periodontology, Department of Restorative Dentistry, Periodontology and Endodontology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany
Search for more papers by this authorCorresponding Author
U. Völker
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Prof. Dr. Uwe Völker, Ernst-Moritz-Arndt-University Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, Friedrich-Ludwig-Jahn-Str. 15a, 17487 Greifswald, Germany
Tel: +49 3834 86 5870
Fax: +49 3834 86 795871
e-mail: [email protected]
Search for more papers by this authorN. Jehmlich
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorK. H. D. Dinh
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorM. Gesell-Salazar
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorE. Hammer
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorL. Steil
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorV. M. Dhople
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorC. Schurmann
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorB. Holtfreter
Unit of Periodontology, Department of Restorative Dentistry, Periodontology and Endodontology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany
Search for more papers by this authorT. Kocher
Unit of Periodontology, Department of Restorative Dentistry, Periodontology and Endodontology, University Medicine Greifswald, Ernst-Moritz-Arndt-University, Greifswald, Germany
Search for more papers by this authorCorresponding Author
U. Völker
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Prof. Dr. Uwe Völker, Ernst-Moritz-Arndt-University Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, Friedrich-Ludwig-Jahn-Str. 15a, 17487 Greifswald, Germany
Tel: +49 3834 86 5870
Fax: +49 3834 86 795871
e-mail: [email protected]
Search for more papers by this authorAbstract
Background and Objective
Interest in human saliva is increasing for disease-specific biomarker discovery studies. However, protein composition of whole saliva can grossly vary with physiological and environmental factors over time and it comprises human as well as bacterial proteins.
Material and Methods
We compared intra- and inter-subject variabilities using complementary gel-based (two-dimensional difference gel electrophoresis, 2-D DIGE) and gel-free (liquid chromatography tandem mass spectrometry, LC-MS/MS) proteomics profiling of saliva. Unstimulated whole saliva of four subjects was examined at three different time-points (08.00 h, 12.00 h and 17.00 h) and variability of the saliva proteome was analyzed on two successive days by LC-MS/MS.
Results
In the 2-D DIGE experiment, the median coefficient of variation (CV) for intra-subject variability was significantly lower (CV of 0.39) than that for inter-subject variability (CV of 0.57; CV of technical replicates 0.17). LC-MS/MS data confirmed the significantly lower variation within subjects over time (CV of 0.37) than the inter-subject variability (CV of 0.53; CV of technical replicates 0.11), and that the inter-subject variability was not time-dependent.
Conclusion
Both techniques revealed similar trends of variations on technical, intra- and inter-subject level but provided peptide and protein focused information and should thus be used as complementary approaches. The data presented indicate that 2-D DIGE as well as LC-MS/MS approaches are suitable for biomarker screening in saliva.
Supporting Information
| Filename | Description |
|---|---|
| jre12025-sup-0001-FigureS1.pdfapplication/PDF, 115.9 KB | Figure S1. For identification of protein spots of whole saliva a pool of 400 μg protein was separated by 2-DE and stained with coomassie brilliant blue. |
| jre12025-sup-0002-FigureS2.pdfapplication/PDF, 95.3 KB | Figure S2. Subject ordered standard deviation of spot volumes of the 2-DIGE experiment. |
| jre12025-sup-0003-FigureS3.pdfapplication/PDF, 95 KB | Figure S3. Subject ordered standard deviation of protein intensities of the LC-MS/MS experiment. |
| jre12025-sup-0004-TableS1.xlsxapplication/Excel, 46 KB | Table S1. MALDI protein identification_n106.xlsx. |
| jre12025-sup-0005-TableS2.xlsxapplication/Excel, 1.7 MB | Table S2. LC–MS protein identification list with corresponding proteins. |
| jre12025-sup-0006-TableS3.xlsxapplication/Excel, 11.1 KB | Table S3. Pearson correlation coefficient (R2) of protein intensities. |
| jre12025-sup-0007-TableS4.xlsxapplication/Excel, 11.1 KB | Table S4. Table of analysis of variance (ANOVA) values of the gel-based and gel-free approaches. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1Spielmann N, Wong D. Saliva: diagnostics and therapeutic perspectives. Oral Dis 2011; 17: 345–354.
- 2Castagnola M, Picciotti PM, Messana I et al. Potential applications of human saliva as diagnostic fluid. Acta Otorhinolaryngol Ital 2011; 31: 347–357.
- 3Hu S, Loo JA, Wong DT. Human saliva proteome analysis and disease biomarker discovery. Expert Rev Proteomics 2007; 4: 531–538.
- 4Punyadeera C, Dimeski G, Kostner K, Beyerlein P, Cooper-White J. One-step homogeneous C-reactive protein assay for saliva. J Immunol Methods 2011; 373: 19–25.
- 5Goncalves LR, Soares MR, Nogueira FC et al. Comparative proteomic analysis of whole saliva from chronic periodontitis patients. J Proteomics 2010; 73: 1334–1341.
- 6Hu S, Loo JA, Wong DT. Human saliva proteome analysis. Ann N Y Acad Sci 2007; 1098: 323–329.
- 7Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011; 57: 675–687.
- 8Giusti L, Baldini C, Bazzichi L et al. Proteome analysis of whole saliva: a new tool for rheumatic diseases–the example of Sjogren's syndrome. Proteomics 2007; 7: 1634–1643.
- 9Lopez ME, Colloca ME, Paez RG, Schallmach JN, Koss MA, Chervonagura A. Salivary characteristics of diabetic children. Braz Dent J 2003; 14: 26–31.
- 10Rao PV, Reddy AP, Lu X et al. Proteomic identification of salivary biomarkers of type-2 diabetes. J Proteome Res 2009; 8: 239–245.
- 11Haigh BJ, Stewart KW, Whelan JR, Barnett MP, Smolenski GA, Wheeler TT. Alterations in the salivary proteome associated with periodontitis. J Clin Periodontol 2010; 37: 241–247.
- 12Dowling P, Wormald R, Meleady P, Henry M, Curran A, Clynes M. Analysis of the saliva proteome from patients with head and neck squamous cell carcinoma reveals differences in abundance levels of proteins associated with tumour progression and metastasis. J Proteomics 2008; 71: 168–175.
- 13Hu S, Arellano M, Boontheung P et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res 2008; 14: 6246–6252.
- 14Xiao H, Zhang L, Zhou H, Lee JM, Garon EB, Wong DTW. Proteomic analysis of human saliva from lung cancer patients using two-dimensional difference gel electrophoresis and mass spectrometry. Mol Cell Proteomics 2012; 11: M111.012112.
- 15Walz A, Stuhler K, Wattenberg A et al. Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis. Proteomics 2006; 6: 1631–1639.
- 16Bandhakavi S, Van RS, Tawfik PN et al. Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva. J Proteome Res 2011; 10: 1052–1061.
- 17Ruhl S. The scientific exploration of saliva in the post-proteomic era: from database back to basic function. Expert Rev Proteomics 2012; 9: 85–96.
- 18Krief G, Deutsch O, Gariba S, Zaks B, Aframian DJ, Palmon A. Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG. Oral Dis 2011; 17: 45–52.
- 19Hardt M, Witkowska HE, Webb S et al. Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents. Anal Chem 2005; 77: 4947–4954.
- 20Ghafouri B, Tagesson C, Lindahl M. Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics 2003; 3: 1003–1015.
- 21Guo T, Rudnick PA, Wang W, Lee CS, Devoe DL, Balgley BM. Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS. J Proteome Res 2006; 5: 1469–1478.
- 22Xie H, Onsongo G, Popko J et al. Proteomics analysis of cells in whole saliva from oral cancer patients via value-added three-dimensional peptide fractionation and tandem mass spectrometry. Mol Cell Proteomics 2008; 7: 486–498.
- 23Bandhakavi S, Stone MD, Onsongo G, Van RS, Griffin TJ. A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva. J Proteome Res 2009; 8: 5590–5600.
- 24Helmerhorst EJ, Oppenheim FG. Saliva: a dynamic proteome. J Dent Res 2007; 86: 680–693.
- 25Esser D, Alvarez-Llamas G, de VM, Weening D, Vonk RJ, Roelofsen H. Sample stability and protein composition of saliva: implications for its use as a diagnostic fluid. Biomark Insights 2008; 3: 25–27.
- 26Quintana M, Palicki O, Lucchi G et al. Inter-individual variability of protein patterns in saliva of healthy adults. J Proteomics 2009; 72: 822–830.
- 27Schulz BL, Cooper-White J, Punyadeera CK. Saliva proteome research: current status and future outlook. Crit Rev Biotechnol 2012; ??: ??–??.
- 28Topkas E, Keith P, Dimeski G, Cooper-White J, Punyadeera C. Evaluation of saliva collection devices for the analysis of proteins. Clin Chim Acta 2012; 413: 1066–1070.
- 29Afkarian M, Bhasin M, Dillon ST et al. Optimizing a proteomics platform for urine biomarker discovery. Mol Cell Proteomics 2010; 9: 2195–2204.
- 30Hammer E, Bien S, Salazar MG et al. Proteomic analysis of doxorubicin-induced changes in the proteome of HepG2cells combining 2-D DIGE and LC-MS/MS approaches. Proteomics 2010; 10: 99–114.
- 31Thiele T, Steil L, Gebhard S et al. Profiling of alterations in platelet proteins during storage of platelet concentrates. Transfusion 2007; 47: 1221–1233.
- 32Steil L, Thiele T, Hammer E et al. Proteomic characterization of freeze-dried human plasma: providing treatment of bleeding disorders without the need for a cold chain. Transfusion 2008; 48: 2356–2363.
- 33Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing. Stat Med 1990; 9: 811–818.
- 34Eymann C, Dreisbach A, Albrecht D et al. A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics 2004; 4: 2849–2876.
- 35Karanam NK, Grabarczyk P, Hammer E et al. Proteome analysis reveals new mechanisms of Bcl11b-loss driven apoptosis. J Proteome Res 2010; 9: 3799–3811.
- 36 R_Development_Core_Team. R: A language and environment for statistical computing, R Development Core Team. Vienna: R Foundation for Statistical Computing, 2008.
- 37Nagaraj N, Mann M. Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome. J Proteome Res 2011; 10: 637–645.
- 38Gusman H, Leone C, Helmerhorst EJ et al. Human salivary gland-specific daily variations in histatin concentrations determined by a novel quantitation technique. Arch Oral Biol 2004; 49: 11–22.
- 39Messana I, Cabras T, Inzitari R et al. Characterization of the human salivary basic proline-rich protein complex by a proteomic approach. J Proteome Res 2004; 3: 792–800.
- 40Millea KM, Krull IS, Chakraborty AB, Gebler JC, Berger SJ. Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry. Biochim Biophys Acta 2007; 1774: 897–906.
- 41Morales-Bozo I, Urztua-Orellana B, Dominguez P, Aguilera S, Lopez-Solis R. Patterns and variability in electrophoretic polypeptide profiles of human saliva in a healthy population. J Physiol Biochem 2006; 62: 179–188.
- 42Neyraud E, Sayd T, Morzel M, Dransfield E. Proteomic analysis of human whole and parotid salivas following stimulation by different tastes. J Proteome Res 2006; 5: 2474–2480.
- 43Neyraud E, Palicki O, Schwartz C, Nicklaus S, Feron G. Variability of human saliva composition: Possible relationships with fat perception and liking. Arch Oral Biol 2012; 57: 556–566.
Citing Literature
