Establishment of a Protocol for CE‐MS Based Peptidome Analysis of Human Saliva

Proteins and peptides indicate physiological or pathological states and are investigated to identify markers to scrutinize health and disease surveillance. Saliva contains many proteins and peptides that could serve as biomarkers, offering a potential noninvasive approach for disease detection. To enable the assessment of the saliva proteome and peptidome as sources of biomarkers, protocols for sampling, sample preparation, and measurements have to be developed. We present the results of peptidome analysis from saliva samples collected at different time points before and after breakfast from 14 healthy adults (50% male, mean age 42.7 ± 10.3 years). While similar methods have been previously applied to urine, our aim was to adapt and demonstrate the effectiveness of these protocols for saliva. Specifically, we aimed to establish a salivary peptide dataspace, including peptide amino acid sequences, and to evaluate the impact of food intake and time of sampling. Capillary electrophoresis-mass spectrometry (CE-MS) and CE-MS/MS were used for peptidome analysis. Per sample, 3147 ± 559 peptides were detectable, without significant differences in the number of detected peptides between sample collection times. However, some peptides differed significantly in their abundance between samples collected before breakfast and 1, 2 and 4 h after breakfast. Samples collected after breakfast were more consistent in their peptide content. Sequencing identified 630 peptides, fragments of 82 proteins, with the majority derived from proline-rich proteins. The data indicate that saliva for peptidomics is best collected 1 to 4 h after breakfast.