title: A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry.
abstract: BACKGROUND
  LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i) to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii) to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii) to evaluate the robustness of biomarkers selection.
  
  
  RESULTS
  A pool of urine sample spiked at increasing concentrations with a mixture of standard peptides was used as a specimen for biological samples with or without biomarkers. Extraction and nano-LC-MS variabilities were estimated by analyzing in triplicates and hexaplicates, respectively. The stability of chromatographic fractions immobilised with MALDI matrix on MALDI plates was evaluated by successive MS acquisitions after different storage times at different temperatures.Low coefficient of variation (CV%: 10-22%) and high correlation (R2 > 0.96) values were obtained for the quantification of the spiked peptides, allowing quantification of these peptides in the low fentomole range, correct group discrimination and selection of "specific" markers using principal component analysis. Excellent peptide integrity and stable signal intensity were found when MALDI plates were stored for periods of up to 2 months at +4 degrees C. This allowed storage of MALDI plates between LC separation and MS acquisition (first decoupling), and between MS and MSMS acquisitions while the selection of inter-group discriminative ions is done (second decoupling). Finally the recording of MSMS spectra to obtain structural information was focused only on discriminative ions in order to minimize analysis time.
  
  
  CONCLUSIONS
  Contrary to other classical approaches with direct online coupling of chromatographic separation and on the flight MS and/or MSMS data acquisition for all detected analytes, our dual decoupling strategy allowed us to focus on the most discriminative analytes, giving us more time to acquire more replicates of the same urine samples thus increasing detection sensitivity and mass precision.
authors.affiliation: INSERM U850, France. khaled.benkali@etu.unilim.fr
authors.forename: K
authors.initials: K
authors.lastname: Benkali
authors.forename: P
authors.initials: P
authors.lastname: Marquet
authors.forename: Jp
authors.initials: J
authors.lastname: Rérolle
authors.forename: Y
authors.initials: Y
authors.lastname: Le Meur
authors.forename: Ln
authors.initials: L
authors.lastname: Gastinel
mesh_terms: D015415:Biomarkers; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D010455:Peptides / Q000652:urine; D025341:Principal Component Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014556:Urine / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D010455:Peptides