High-Sensitivity NanoLC−MS/MS Analysis of Urinary Desmosine and Isodesmosine

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based crosslinker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D 4 -DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 µL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 ± 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 ± 3.1 ng/ mg crea), (c) healthy smokers (13.2 ± 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 ± 2.9 ng/ mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.Chronic obstructive pulmonary disease, or COPD, is a respiratory disorder generally caused by smoking. 1-3 Two major forms of COPD are emphysema and chronic bronchitis. 3 COPD is characterized by impaired lung function, inflammation and scarring of bronchial tubes, and degradation of elastin-containing tissues. The elastin is a major protein component of insoluble fibers providing elastic properties to tissues such as lung, skin, and blood vessels. 4 Also, elastin peptides were reported to be chemotactic for neutrophils and macrophages and could play a role in the progression of pulmonary emphysema once elastin degradation is initiated. 5,6 Most of the COPD biomarkers reported in the literature are cross-linkers 1,7,8 released during the elastin breakdown. The two most abundant and studied elastin crosslinkers are desmosine (DES) and isodesmosine (IDES) 9,10 ( Figure 1). For simplicity, in this report DESs refer to both DES and IDES. DESs are pyridinium-based structural isomers arising from the

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

“…This is because longPEG-DES contrary to shortPEG-DES and medium-PEG-DES can accommodate an extra proton within the PEG chain triggering the typical CID fragmentation of PEG molecules [19,36]. Previous ECD studies on free PEG molecules reported a similar fragmentation pattern as in CID (loss of ethylene oxide units, 44 Da) [14,30]. This type of fragmentation was, however, not observed for any of the studied DES-PEG derivatives upon ETD.…”

“…Derivatization facilitates the analysis by reversed-phase chromatography since it converts a highly hydrophilic analyte into a more hydrophobic compound that is retained on standard reversed phase LC columns [30]. Derivatization results in the conversion of primary amines into amide groups thus modifying the protonation site of the molecule, which may affect the ETD and/or CID patterns.…”

“…DES/IDS were derivatized with polyethyleneglycol (PEG) based on our finding that derivatization of amino acids with PEG increases ionization efficiency by a factor 10-500 ( [29] and unpublished results). In addition, DES/IDS were derivatized with propionic anhydride, as this approach has been employed for the analysis of DES/IDS in urine [30].…”

Fingerprinting Desmosine-Containing Elastin Peptides

“…Derivatization with propionic anhydride was carried out according to Boutin et al [30]: 100 μL of 0.1 N NH 4 HCO 3 and 100 μL of a solution containing 23% (v/v) propionic anhydride and 9% (v/v) NH 4 OH were added to DES and IDES, respectively, and the samples were incubated at room temperature for 1 h. Further derivatives of DES and IDES were prepared using amine 9-fluorenylmethyl chloroformate (FMOC; reacting at amine groups), butanol (reacting at the carboxylic acid groups), and propyl chloroformate (PrCl; reacting with both types) according to Uutela et al [31]. All samples were evaporated to dryness using a centrifugal evaporator and then dissolved in 1 mL of MeOH/H 2 O (80:20, v/v).…”

Development of a Robust LC-MS/MS Method for Determination of Desmosine and Isodesmosine in Human Urine

“…In recently study, Boutin et al have reported a high sensitive analytical method using nanofl ow liquid chromatography tandem mass spectrometry nanoLC-MS/MS , after the sample derivatization 10 . None of these studies have suggested the robustness of their methods as they have evaluated only the reproducibility and the recovery of the methods.…”