Benkali K, Marquet P, Rérolle Jp, Le Meur Y, Gastinel Ln
INSERM U850, France.
BMC Genomics. 2008 Nov 14;9:541. doi: 10.1186/1471-2164-9-541.
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.
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.
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.
液相色谱 - 基质辅助激光解吸电离飞行时间串联质谱（LC - MALDI - TOF/TOF）分析是生物标志物发现中的一种强大工具，具有高灵敏度和高通量能力。然而，基于MALDI - TOF/TOF的生物标志物发现方法仍需优化，特别是要减少分析时间并评估其峰强度测量的重现性。本方法学研究的目的是：（i）优化并严格评估基于纳升液相色谱（Nano - LC）离线耦合到MALDI - TOF/TOF的尿液生物标志物发现方法的每个步骤，充分利用Nano - LC、质谱（MS）和串联质谱（MS/MS）之间的双重解耦来减少整体分析时间；（ii）评估纳升液相色谱 - MALDI分析在生物标志物发现中的定量性能和重现性；（iii）评估生物标志物选择的稳健性。
用标准肽混合物以递增浓度加标的尿液样本池用作有或无生物标志物的生物样本的标本。分别通过三次重复和六次重复分析来估计提取和纳升液相色谱 - 质谱的变异性。通过在不同温度下不同储存时间后连续进行质谱采集，评估了用MALDI基质固定在MALDI板上的色谱馏分的稳定性。对于加标肽的定量，获得了低变异系数（CV%：10 - 22%）和高相关性（R2 > 0.96）值，从而能够在低飞摩尔范围内对这些肽进行定量，使用主成分分析进行正确的组区分和“特异性”标志物的选择。当MALDI板在4℃下储存长达2个月时，发现肽的完整性良好且信号强度稳定。这允许在液相色谱分离和质谱采集之间（第一次解耦）以及在质谱和串联质谱采集之间（在进行组间鉴别离子选择时，第二次解耦）储存MALDI板。最后，记录串联质谱谱图以获得结构信息仅聚焦于鉴别离子，以尽量减少分析时间。
与其他将色谱分离直接在线耦合以及对所有检测到的分析物进行飞行中质谱和/或串联质谱数据采集的经典方法不同，我们的双重解耦策略使我们能够专注于最具鉴别力的分析物，从而有更多时间对相同尿液样本进行更多次重复分析，进而提高检测灵敏度和质量精度。
