Identification of biomarkers for endometriosis using clinical proteomics

Abstract

Background: We investigated possible biomarkers for endometriosis (EM) using the ClinProt technique and proteomics methods.

Methods: We enrolled 50 patients with EM, 34 with benign ovarian neoplasms and 40 healthy volunteers in this study. Serum proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) combined with weak cationic exchange (WCX) magnetic beads. Possible biomarkers were analyzed by a random and repeat pattern model-validation method that we designed, and ClinProtools software, results were refined using online liquid chromatography-tandem MS.

Results: We found a cluster of 5 peptides (4210, 5264, 2660, 5635, and 5904 Da), using 3 peptides (4210, 5904, 2660 Da) to discriminate EM patients from healthy volunteers, with 96.67% sensitivity and 100% specificity. We selected 4210 and 5904 m/z, which differed most between patients with EM and controls, and identified them as fragments of ATP1B4, and the fibrinogen alpha (FGA) isoform 1/2 of the FGA chain precursor, respectively.

Conclusions: ClinProt can identify EM biomarkers, which - most notably - distinguish even early-stage or minimal disease. We found 5 stable peaks at 4210, 5264, 2660, 5635, and 5904 Da as potential EM biomarkers, the strongest of which were associated with ATP1B4 (4210 Da) and FGA (5904 Da); this indicates that ATP1B4 and FGA are associated with EM pathogenesis.

Figure 1

Protein standard fingerprints in different experimental runs. (a) Six proteins in different experimental runs; (b) Entire standard fingerprint in different experimental runs. Blue: 1^st run; red: 2^nd run; green: 3^rd run; purple: 4^th run.

Figure 2

Distribution of endometriosis (EM) and control. X: EM samples; O: Control samples.

Figure 3

Differential protein peaks in typical samples, and in electrophoresis gel bands. Left: Differential protein peaks; red: Patient samples; green: Healthy controls; Right: Electrophoresis gel bands; Upper: Healthy controls; lower: Patients. (a) 2660 Da; (b) 4210 Da; (c) 5264 Da; (d) 5635 Da; and (e) 5904 Da.

Figure 4

Peptide mass fingerprinting of (a) 4210 m/z; (b) 5904 m/z; (c) 5264 m/z; (d) 5635 m/z; and (e) 2600 m/z.

Figure 5

Distribution and performance of serum peptide biomarker for (a) 4210 m/z and (b) 5904 m/z. Receiver operator characteristic of (c) 4210 m/z and (d) 5905 m/z distinguishes patients with endometriosis (EM) from healthy controls. Red: Controls; green: Patients with EM.