Mass spectrometry strategies applied to the characterization of proline‐rich peptides from secretory parotid granules of pig (Sus scrofa)

Abstract

Basic proline-rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline-rich peptides (SP-A and SP-B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss-Prot data bank: Q95JC9-1, Q95JC9-2 and Q95JC9-3). Together the coding regions for SP-A and SP-B, which are repeated many times, account for 52-70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP-HPLC-ESI-IT-MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline-rich peptides derived from the three proproteins. Together with the coding regions for SP-A and SP-B already identified it was possible to assign 68-75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases.

Micro-heterogeneity of human saliva peptide P-C characterized by high-resolution top-down fourier-transform mass spectrometry

“…This ion at m/z 1066.0447 was later assigned to the b21-H 2 O product ion of the lighter form of Peptide P-C (Variant 1). At the same time, the isotopomer cluster for the b15 IRMPD product ion presented an overabundance of the first 13 C component, indicating the presence of a species 1 Da heavier than the monoisotopic peak (data not shown). These features led to further investigations of the possible presence of two variants of the peptide that differ by the nominal mass of Ϯ 1Da.…”

“…Peptide P-C was placed at 37°C directly from the HPLC fraction (ϳ20% acetonitrile, acidic pH) or re-dissolved in ammonium bicarbonate (pH 7.5) after drying. CAD MS/MS spectra recorded over the following 10 days revealed that the relative intensity of the 13 C peak was unchanged, allowing conclusion that the heavier form likely corresponds to a SNP rather than a deamidation process. In addition, the likelihood of such deamidation process occurring during sample handling/processing was deemed relatively low because the samples were not exposed to basic pH [24,25].…”

“…This is attributed to misassignment of monoisotopic versus 13 C peaks for the regular and Variant 2 (deamidated/ SNP) forms as a result of incomplete peak lists for the minor sequence variant.…”

“…This is assigned this to the fact that the Xtract software could not all three distributions, and thus incorrect peaks (e.g., 13 C of the regular Peptide P-C instead of the monoisotopic peak of the deamidated form) were used for assignments by the ProSight software. A solution to this problem lies in the modeling of theoretical profiles to better match data.…”

“…By supplementing low-resolution intact mass measurements obtained on quadrupole instruments with MS/MS data after proteolysis, a good start was made toward documentation of the processing and modifications of several classes of salivary proteins (e.g., histatin and statherin), and their variability between individuals [3,7,[11][12][13]. More recently this approach was successfully used by Messana et al to elucidate saliva protein trafficking in concert with post-translational events such as phosphorylation, sulfation, and cleavage of propeptides [14].…”