Peptidomics: Identification and quantification of endogenous peptides in neuroendocrine tissues

Abstract

Neuropeptides perform a large variety of functions as intercellular signaling molecules. While most proteomic studies involve digestion of the proteins with trypsin or other proteases, peptidomics studies usually analyze the native peptide forms. Neuropeptides can be studied by using mass spectrometry for identification and quantitation. In many cases, mass spectrometry provides an understanding of the precise molecular form of the native peptide, including post-translational cleavages and other modifications. Quantitative peptidomics studies generally use differential isotopic tags to label two sets of extracted peptides, as done with proteomic studies, except that the Cys-based reagents typically used for quantitation of proteins are not suitable because most peptides lack Cys residues. Instead, a number of amine-specific labels have been created and some of these are useful for peptide quantitation by mass spectrometry. In this review, peptidomics techniques are discussed along with the major findings of many recent studies and future directions for the field.

Substrate Specificity of Human Carboxypeptidase A6

“…Different amounts of purified CPA6 enzyme were incubated with a peptide mixture extracted from mouse brain, representative of the peptidome of the mouse brain that might be encountered by secreted CPA6. After incubation with enzyme, the peptides were differentially labeled with isotopic tags, combined, and analyzed by liquid chromatography/mass spectrometry (LC-MS) (23,35,36). Over 100 peptides were detected through these LC-MS analyses, with close to 50 being identified through a combination of tandem mass spectrometry (MS/MS) and close matches with previously identified peptides; the criteria used for the matches included an observed monoisotopic mass within 0.004% of the theoretical mass, an expected charge equal to the number of basic residues plus the N terminus, and a correct number of isotopic tags incorporated.…”

Quantification of Polypeptides by Mass Spectrometry

“…Compounds such as N-acetoxysuccinimide [70], succinic anhydride [70], acetic anhydride [71], and trimethylammonium chloride [72] have been successfully used to label amines in polypeptides as the basis for quantitative peptidomic analyses. In essence, any amine-reactive compound can be used to label peptides, the limitation being whether these reagents can be synthesized by incorporating stable isotopes and their stability under storage [2]. In each case and in analogy to the ICAT approach, this strategy for quantitative peptidomics involves labeling endogenous or protein-derived peptides, which are then mixed and the resultant mixture analyzed by LC-MS. As with the ICAT strategy, quantification is achieved by comparing the ion intensities of peptides incorporating heavy and light isotopes in MS spectra, while identification is achieved by switching from MS to MS/MS modes in data-dependent acquisition experiments.…”

Recent advances of mesoporous materials in sample preparation

“…Peptidomics, referring to all of LMW peptides or proteins, is defined as the systematic analysis of endogenous peptides and small proteins in biological sample (such as body fluids, cell lysate, and tissue extract) at a defined time points and/or locations [58][59][60]. Peptides from these biological samples may have the potential to serve as biomarkers, indicating of progression from a normal to a diseased status [61].…”