Capillary Electrophoresis and Top Down ESI FT-ICR Mass Spectrometry for the Characterization of Renal Disease Biomarkers

Introduction

End stage renal diseases (ESRD), such as IgA nephropathy and focal segmental glomerulosclerosis (FSGS) typically result in the loss of the kidney, unless diagnosed and treated early. Associated with ESRD progression is the occurrence of proteinuria (an increase in the protein content of the urine). Statistical analysis of CE-ESI ToF MS data acquired from the urine of healthy and diseased patients yields multiple biomarkers of renal damage. We have demonstrated that capillary electrophoresis mass spectrometry (CE-ToF MS) detection of these markers is an effective method for the diagnosis of renal disease, and offers a non-invasive alternative to biopsy. The resolution, mass accuracy, and multiple modes of ion fragmentation make top-down FT-ICR MS/MS an attractive method for the characterization of these biomarkers, many of which are >8 kDa.

Methods and Instrumentation

Urine samples were collected and prepared as described elsewhere [1]. CE (Crystal 310, Thermo Electron Corp.) was performed off-line with fraction collection achieved with a HTS PAL (LEAP technologies) fraction collector and on-line, interfaced to a homebuilt 9.4T FT-ICR instrumentwith a liquid sheath ESI interface (Agilent). CE ESI MS conditions were as described previously [1]. Multiple replicate fractions acquired off-line were pooled and transferred to a chip based nanoESI device (Nanomate, Advion) to provide extended analysis time. Product ion data was obtained by external quadrupole isolation of the precursor ion followed by IRMPD and/or ECD MS/MS.

Preliminary Data

Hundreds of CE ESI ToF MS experiments followed by data reduction (mosaiqueVisu software, Mosaique Diagnostics) and statistical analysis (see [2] for details) have established new biomarkers of FSGS with high specificity and selectivity. Initial CE FT-ICR MS experiments, performed on-line, accurately determined the precursor ion m/z ratio of a number of interesting FSGS biomarkers that were targeted for MS/MS analysis. These compounds were then identified in the off-line fractions and IRMDP and/or ECD MS/MS was used to generate product ion data. The combination of CE with on-line and off-line analysis allows for experimental validation with different mass spectrometers and extends MS/MS analysis time. This approach allowed the identification of five new biomarkers (2-10 kDa) for FSGS. The identifications are unambiguous, relate directly to the diagnostic ions in the clinical ESI ToF MS assay, and could not have been achieved without the use of preparative CE followed by top-down FT-ICR MS/MS.