Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry
- 1 June 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 21 (6), 908-917
- https://doi.org/10.1016/j.jasms.2010.01.025
Abstract
A hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm, allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA, and SN were cleaved of a N-terminal 20 amino acid signal peptide and cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, cystatin D isoforms with 24 and 28 amino acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3, in agreement with previous work. Both cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14,328 Da isoform of cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. With fully characterized cystatin intact mass tags it will now be possible to examine the correlation between the abundance of these molecules and human health and disease.Keywords
This publication has 36 references indexed in Scilit:
- Age-Dependent Modifications of the Human Salivary Secretory Protein ComplexJournal of Proteome Research, 2009
- Cystatin-C is an independent prognostic factor for survival in multiple myeloma and is reduced by bortezomib administrationHaematologica, 2009
- Potential biomarkers of human salivary function: A modified proteomic approachArchives of Oral Biology, 2009
- Hypo-Phosphorylation of Salivary Peptidome as a Clue to the Molecular Pathogenesis of Autism Spectrum DisordersJournal of Proteome Research, 2008
- Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us?Journal of Separation Science, 2008
- Trafficking and Postsecretory Events Responsible for the Formation of Secreted Human Salivary PeptidesMolecular & Cellular Proteomics, 2008
- The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal SecretionsJournal of Proteome Research, 2008
- Protein-sequence polymorphisms and post-translational modifications in proteins from human saliva using top-down Fourier-transform ion cyclotron resonance mass spectrometryInternational Journal of Mass Spectrometry, 2007
- Human glandular salivas: their separate collection and analysisEuropean Journal of Oral Sciences, 1996
- The disulphide bridges of human cystatin C (γ-trace) and chicken cystatinFEBS Letters, 1984