Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry

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.

Figure 1. Reversed-phase chromatography of human salivary proteins

Whole saliva, as well as parotid and sub-mandibular/sub-lingual ductal secretions were dried and dissolved in 6M guanidine prior to separation by HPLC and online ESI mass spectrometry with fraction collection (LC-MS+; see methods). Typical total ion chromatograms are shown highlighting the region where the Cystatin family elutes. Intact Cystatins were found in only whole saliva and SMSL secretions.

Figure 2. Top-down mass spectrometry of Cystatins S, S1, and S2

Appropriate fractions were analyzed by static nanospray on a 7 T LTQFT Ultra. CAD experiments on isolated precursor ions yielded complex tandem mass spectra that were interpreted using Prosight PC software (see methods). The precursor ion isolation used for each experiment is shown on the left (m/z) while the b- and y- product ion assignments observed at 10 ppm tolerance (deltamass feature deactivated) are shown superimposed on the sequence to the right. A twenty amino-acid signal peptide has been removed from the precursor sequence and each Cys residue has been oxidized (−1.0078 Da) to reflect the intact pair of disulfide bonds of the native protein. For Cystatin S 41% of product ions were matched to b- and y- ions giving a Pscore of P-Score of 1.71E-57. For Cystatin S1 a single phosphorylation (+79.9663 Da) was added at position 3 of the mature protein with 31% of product ions matched and a Pscore of 4.75E-42. For Cystatin S2 a second phosphorylation was added at position 1 of the mature protein with 26% of product ions matched and a Pscore of 8.06E-32.

Figure 3. Top-down mass spectrometry of Cystatins SA, SN and C

Appropriate fractions were analyzed as in Figure 2. For Cystatin SA and SN a twenty amino-acid signal peptide was removed while for Cystatin C a twenty-six amino-acid peptide was removed, conserving the N-terminus of the mature protein. Each Cys residue has been oxidized (−1.0078 Da) to reflect the intact pair of disulfide bonds of the native protein. For Cystatin SA 34% of product ions were matched giving a Pscore of 2.8E-46. For Cystatin SN 30% of product ions were matched giving a Pscore of 4.3E-29. For Cystatin C 22% of product ions were matched giving a Pscore of 1.25E-34.

Figure 4. Top-down mass spectrometry of two isoforms of Cystatin D

A. A protein of average mass 13605 Da that was not detected by LC-MS+ was analyzed by top-down CAD of the 13+ precursor ion (inset top right) giving the product ion spectrum shown. The precursor and product ion masses were accounted for by removing 24 amino-acids from the N-terminus of the human Cystatin D sequence, oxidizing the 4 Cys residues to reflect two disulfide bonds and making the C46R polymorphism arising from a known SNP (Table 1). 31% of product ions were matched giving a Pscore of 3.44E-18. B. A protein of average mass 13165 Da that was detected by LC-MS+ was analyzed by top-down CAD of the 11+ precursor ion (inset top right) giving the product ion spectrum shown. The precursor and product ion masses were accounted for by removing 28 amino-acids from the N-terminus of the human Cystatin D sequence, oxidizing the 4 Cys residues to reflect two disulfide bonds and making the C46R polymorphism arising from a known SNP (Table 1). 51% of product ions were matched giving a Pscore of 2.0E-22. A small section of the product ion spectrum was expanded to show unique b-ions (top left in A and B).

Figure 5. Sequence alignment of the abundant human salivary Cystatins

ClustalW was used for the alignment [36].