Size-selective extraction of peptides from urine for mass spectrometric analysis.

Abstract

Protein excretion in urine has been suggested as an indicator of kidney disease since the time of Hippocrates. In the early 1800s, Bright further established approaches for studying proteinuria as a marker for kidney disease (1). As methods for quantitative and qualitative analysis have become more sophisticated, it has become possible to detect earlier stages of kidney disease and to differentiate different patterns of protein excretion (1)(2)(3). Quantitative immunoassays of selected urinary components such as α1-microglobulin, albumin, IgG, and α2-macroglobulin have been shown to be useful in characterizing the nature of proteinuria (4). Two-dimensional electrophoresis has provided a method for simultaneous analysis of numerous proteins in urine (5)(6). Recently, a new dimension has been added to analysis of urinary components by mass spectrometric techniques, which detect many small peptide components below the size resolution of electrophoresis (7)(8). The highly complex mixtures of small peptides in urine offer the potential for information-rich patterns for clinical diagnosis. Concentrations of urinary peptides serve not only as markers for kidney function but also as markers of other systemic physiologic processes. As examples, immunoassays for specific peptides provide measures of thrombosis and fibrinolysis (9)(10) and endocrine function (11). In the present study, we sought to identify a simple method to prepare urine specimens for the analysis of small peptide components. Sample preparation represents one of the major challenges for analysis of peptide components in urine specimens by mass spectrometry. Ideally, sample preparation needs to accomplish three tasks: (a) concentration of relatively dilute peptide components; (b) removal of salts that suppress peptide ionization in mass spectrometry; and (c) depletion of albumin and other high-molecular-weight components that comprise most of the total protein mass in urine. Standard methods that have been applied …