Fingerprinting Desmosine-Containing Elastin Peptides

Abstract

Abstract. Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS 2 data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type C x H y N, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

A comprehensive map of human elastin cross‐linking during elastogenesis

“…Tetrafunctionally cross-linked peptides were identified by a recently developed liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method based on the release of reporter ions [23] from DES/ IDES structures upon dissociation at elevated collision energies. DES and IDES are not distinguishable by this method since they release the same abundant lowmass fragments.…”

“…Each precursor ion was fragmented twice: 1) in stepped HCD mode (29% AE 10% NCE) or CID mode (35% NCE) to obtain sequence information and 2) with 95% NCE to release DES/IDES reporter ions [23]. Peptides were analyzed using the abovementioned solvents by applying an ACN gradient from 1% to 35% (v/v) within 120 min.…”

“…Tetrafunctionally cross-linked peptides were discovered based on their released reporter ions [23] upon fragmentation with elevated collision energies (95% NCE). The presence of at least 12 out of the 15 major reporter ions was set as threshold for a positive hit.…”

“…1). We therefore developed the recently introduced mass spectrometric and bioinformatics methods for analyzing covalent cross-links of mature elastin [22,23]. Another bifunctional cross-link, allysine aldol (AA), is formed by aldol condensation of two Lya moieties [16].…”

Unique molecular networks: Formation and role of elastin cross‐links

“…Elastases including some serine, cysteine, and metalloproteases serve this purpose, and cross‐linking sites can be identified by analyzing the released cross‐linked peptides. While peptide sequences and cross‐link locations have been determined in the past with classical methods such as Edman degradation, more recent studies utilized sensitive high‐resolution mass spectrometric (MS) analyses in combination with customized software . Bifunctional intradomain cross‐links release internally cross‐linked peptides with a cyclic structure upon hydrolysis, whereas bi‐ and higher functional interdomain cross‐links are released as interconnected peptide species, which are challenging with respect to their analyses.…”