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. 2009 Mar 1;81(5):1881-7.
doi: 10.1021/ac801745d.

High-sensitivity nanoLC-MS/MS Analysis of Urinary Desmosine and Isodesmosine

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Free PMC article

High-sensitivity nanoLC-MS/MS Analysis of Urinary Desmosine and Isodesmosine

Michel Boutin et al. Anal Chem. .
Free PMC article

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 cross-linker 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 microL 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.

Figures

Figure 1
Figure 1
Structure of (A) desmosine (DES) (X,R = H); deuterated desmosine (D4-DES) (X = d, R = H); propionylated desmosine (DES(prop)) (X = H, R = COCH2CH3), and (B) isodesmosine (IDES) (R = H); propionylated isodesmosine (IDES(prop)) (R = COCH2CH3).
Figure 2
Figure 2
NanoLC—MS analysis of 25 ng of DES standards propionylated in methanol (MeOH) (A–D) and in isopropanol (i-Pr) (E–H). (A and E) Base peak chromatogram (BPC) (m/z 650–800). (B and F) Reconstructed ion chromatogram (RIC) of DES(prop) (m/z 750). (C and G) RIC of DES(prop) esterified with MeOH (m/z 764) and i-Pr (m/z 792), respectively. (D and H) RIC of DES(prop) amidated (m/z 749).
Figure 3
Figure 3
NanoLC—MS analysis of DES(prop) in urine. (A) Total ion chromatogram (TIC) of the urine sample using the enhanced MS (EMS) mode (m/z 400–1600), part B) Reconstructed ion chromatogram (RIC) corresponding to DESs(prop) (m/z 750.4) in the urine sample, (C) MS spectrum of urine at the elution time of DESs(prop) (9.74 min). A 50 µL urine sample was digested in HCl, purified on an MCX cartridge, and propionylated. See Experimental Section for conditions.
Figure 4
Figure 4
Tandem mass spectrometry analyses of (A) DES(prop) and (B) D4-DES(prop). Product ion spectra were optimized for the transitions corresponding to the lost of one propionyl group (m/z 694 and 698, respectively). Coll. gas, 12; Ecoll, 58 eV.
Figure 5
Figure 5
Box-whisker plot of the DESs concentrations measured for the four groups of urine samples. Box lines indicate lower and higher quartile values with the median as dotted bar. The whiskers show the smallest and largest nonoutlier DESs concentrations. Mean and relative square deviations are presented in brackets for each group.

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