Comparative Urine Analysis by Liquid Chromatography-Mass Spectrometry and Multivariate Statistics: Method Development, Evaluation, and Application to Proteinuria

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• 作者：Ramses F. J. Kemperman ; Peter L. Horvatovich ; Berend Hoekman ; Theo H. Reijmers ; Frits A. J. Muskiet ; Rainer Bischoff
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：January 2007
• 年：2007
• 卷：6
• 期：1
• 页码：194 - 206
• 全文大小：483K
• 年卷期：v.6,no.1(January 2007)
• ISSN：1535-3907

文摘

We describe a platform for the comparative profiling of urine using reversed-phase liquid chromatography-mass spectrometry (LC-MS) and multivariate statistical data analysis. Urinary compounds wereseparated by gradient elution and subsequently detected by electrospray Ion-Trap MS. The lower limitof detection (5.7-21 nmol/L), within-day (2.9-19%) and between-day (4.8-19%) analytical variation ofpeak areas, linearity (R²: 0.918-0.999), and standard deviation for retention time (<0.52 min) of themethod were assessed by means of addition of seven 3-8 amino acid peptides (0-500 nmol/L). Relatingthe amount of injected urine to the area under the curve (AUC) of the chromatographic trace at 214 nmbetter reduced the coefficient of variation (CV) of the AUC of the total ion chromatogram (CV = 10.1%)than relating it to creatinine (CV = 38.4%). LC-MS data were processed, and the common peak matrixwas analyzed by principal component analysis (PCA) after supervised classification by the nearestshrunken centroid algorithm. The feasibility of the method to discriminate urine samples of differingcompositions was evaluated by (i) addition of seven peptides at nanomolar concentrations to blankurine samples of different origin and (ii) a study of urine from kidney patients with and withoutproteinuria. (i) The added peptides were ranked as highly discriminatory peaks despite significantbiological variation. (ii) Ninety-two peaks were selected best discriminating proteinuric from nonproteinuric samples, of which 6 were more intense in the majority of the proteinuric samples. Two ofthese 6 peaks were identified as albumin-derived peptides, which is in accordance with the early riseof albumin during glomerular proteinuria. Interestingly, other albumin-derived peptides were nondiscriminatory indicating preferential proteolysis at some cleavage sites.