Endogenous peptides can be regarded as versatile biomarkers because:
- Peptides are capable to reflect changes in the expression of their precursor proteins
- Peptides are mirroring activity of the corresponding processing proteases
- Peptides have a relatively low molecular weight, that increases the susceptibility to peripheral detection
The term peptide is typically used for polypeptides in the range up to approximately 20 kDa molecular weight. The origin of the term “peptide” (derived from the Greek terms „ peptos”, meaning digestible, and “poly-”, referring to its composition of two or more amino acids) reflects the fact that peptides are usually the products of proteolytic cleavage events . Many pathological processes are associated with changes in protease expression or activation and these changes are captured or represented in altered peptide profiles. Peptidomics® is defined as the systematic, comprehensive, qualitative and quantitative multiplex (e.g. mass spectrometry) analysis of endogenous peptides in a biological sample.
Samples are separated by means of RP-HPLC (reversed phase high pressure liquid chromatography, A) and peptides eluting from the HPLC column are collected into 96 fractions. Each fraction is subjected to MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, B) and the mass spectra of all 96 fractions are combined, resulting in an in silico two-dimensional display of peptide masses, where the x-axis displays the mass-to-charge ratio, the y-axis is determined by the retention time on the RP-HPLC or the fraction number and signal intensity is depicted by color saturation (C). The Spectromania software allow for the processing and analysis of vast amounts of mass spectra and adjacent metadata. Mass spectra of individual samples can be superimposed or correlated against metadata/surrogates, facilitating the visualization and detection of differences in the resultant peptide display (D). For the identification of peptides, peaks from individual HPLC fractions can be subjected to nESI-qTOF-MS/MS or MALDI-TOF/TOF-MS sequencing, resulting in peptide fragment spectra (E). These spectra serve to identify the corresponding peptide sequence by remote database searching.