Objective: The primary objective of the study is the comparison of protein concentrations between patients with stress urinary incontinence (SUI) and healthy controls.
Aim: This pilot study aims to determine a possible altered protein profile in women suffering from SUI, compared to healthy women and therefore to discriminate a potential protein biomarker for SUI.
Main outcome measure: Concentration of each protein in the urine
Trial design: Prospective case- control study
Setting: Department of Gynecology and Obstetrics of the Medical University of Vienna in co- operation with the Core Facilities Proteomics of the Medical University of Vienna
Population: Twenty female patients with SUI and twenty healthy patients who attend the outpatient clinic of the Department of Obstetrics and Gynaecology, Medical University of Vienna (sample size calculation FDR 0.05, power of 80%, assumed proportion of true H0 0.95, assumed standardized effect size of 1)
Methods: After written informed consent, the following examinations are carried out: patient history, provocative stress test, ICIQ short form questionnaire, residual urine volume (ultrasound) and urine analysis (dipstick testing). The patient is asked to deliver a urine sample in a sterilized measure cup labeled with the patients' code, to which proteinase will be added immediately. Two serum blood vials (each 9ml) are taken from a peripheral vein of the patient for routine laboratory and further research. The women of the healthy control group will be recruited from the gynaecological outpatient clinic and will be matched with the case group regarding the age.
Proteomics analysis of urine samples obtained from patients suffering stress urinary incontinence and the control group will be performed using chromatographic separation (LC) with mass spectrometric detection (MS). Due to the heterogeneity of proteins present in urine, it is possible that multidimensional separation of tryptically digested proteins is needed.
After sample collection, urine will be stored at 4°C and filtered on the same day. Filtered urine will be frozen at -80°C until sample preparation and analysis. Sample preparation and analysis will include:
1. Sample collection and immediate addition of protease inhibitor cocktail (Roche, Complete Protease Inhibitor Cocktail).
2. Urine centrifugation and filtration for removal of cell debris.
3. Protein precipitation by applying methanol/chloroform separation for removal of all possible non-proteinic substances.
4. Protein digestion applying in-solution trypsin, pepsin, and Glu-C.
5. Peptide separation using nano HPLC and different chromatographic approaches.
a. Reversed phase (RP) separation of peptides and MS detection
b. Separation of peptides using HILIC (hydrophilic Interaction Liquid Chromatography) for discrimination of polar peptides.
c. In addition to RP and HILIC directly coupled to MS, digested peptides will be separated using multidimensional approaches. Weak anion exchange columns operated under HILIC conditions will be used to separate peptides carrying posttranslational modifications such as phosphorylation or acetylation, thus, increasing the dynamic range of detection. During this separation, fractions will be collected and re-injected onto the RP and HILIC with MS detection.
d. All nano HPLC separations will be performed using biocompatible separation system.
Mass spectrometric analysis of digested peptides will be performed using two different detection methods: ion-trap and the time-of-flight (qToF) MS. qToF mass spectrometric detection and analysis will also be used for the label-free quantitation of peptides and proteins detected in samples. All measurements will be performed in triplicate to provide corrections for technical variability of separation and ionization.
General database search will be performed using the Human SwissProt Database in its actual version at the time of analysis. Data search will be performed using Mascot (http://www.matrixscience.com/) and X!Tandem (http://www.thegpm.org/tandem/) search machines, and the final data allocation and filtering by using Scaffold (www.proteomsoftware.com).