Identification of the human salivary cystatin complex by the coupling of high‐performance liquid chromatography and ion‐trap mass spectrometry

Abstract

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

“…1. Characterization of these proteins, mainly based on the RP-HPLC-ESI-MS and Tandem/MS analysis, has been previously described by some of us (13)(14)(15)(16)(17)(18)(19)(20)(21). Cystatin C and its sulfoxide derivative on methionine 14 were confirmed in the present study by high resolution HPLC-ESI-MS/MS analysis of trypsin digestion products and data are reported in supplemental Table S1.…”

“…Characterization of Salivary Peptides and Proteins-Proteins (salivary acidic proline-rich proteins, histatins, salivary cystatins, statherin, proline-rich peptide P-B, ␣-defensins 1-4, cystatins A, B, C, beta-thymosins (4 and 10), S100A7 (D27), S100A8, S100A9, and S100A12 proteins) and several derivatives (acetylated, cysteinylated, glutathionylated, phosphorylated, and oxidized forms) have been already identified in our previous studies (13)(14)(15)(16)(17)(18)(19)(20)(21). Experimental mass values were obtained by deconvolution of averaged ESI-MS spectra automatically performed using MagTran 1.0 software (22).…”

Human body fluid proteome analysis

“…Both FFE and CIEF were successfully coupled with LIT-MS analysis, leading to the identification of 437 and 1381 proteins, respectively, in whole saliva [373,374]. In addition, a specific subgroup of saliva proteins such as proline-rich proteins (PRPs), histatins, and cystatins, were independently identified and characterized [375][376][377][378], and relative quantitation of saliva LMW peptides was also demonstrated using isotope tagging for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF [379].…”

Trafficking and Postsecretory Events Responsible for the Formation of Secreted Human Salivary Peptides

“…By RP 1 -HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva we have identified in the chromatographic pattern more than 120 different proteins and naturally occurring peptides (1)(2)(3)(4)(5)(6). Their characterization was performed by a variety of mass spectrometric techniques coupled with different enzymatic treatments and amino acid sequencing.…”

“…This study describes the comparative profiling of intact proteins and naturally occurring peptides of cellular fractions enriched in secretory granules (enriched granule preparations (EGPs)) as well as parotid (Pr), submandibular/sublingual (Sm/ Sl), and whole saliva by the above cited RP-HPLC-ESI-MS methods (1)(2)(3)(4)(5)(6). The results provide information useful for a better understanding of the spatial and temporal relationships of the modifications the proteins undergo from their synthesis on the ribosomes to their isolation from whole saliva.…”