Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry.
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.
Ryan
Christopher M
CM
The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90024, USA.
Souda
Puneet
P
Halgand
Frederic
F
Wong
David T
DT
Loo
Joseph A
JA
Faull
Kym F
KF
Whitelegge
Julian P
JP
D000595:Amino Acid Sequence; D056148:Chromatography, Reverse-Phase; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D012463:Saliva / Q000737:chemistry*; D055333:Salivary Cystatins / Q000737:chemistry* / Q000235:genetics; D012469:Salivary Glands / Q000378:metabolism; D016415:Sequence Alignment
D020033:Protein Isoforms; D055333:Salivary Cystatins