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Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples.

Author(s): Lafitte D, Dussol B, Andersen S, Vazi A, Dupuy P, Jensen ON, Berland Y, Verdier JM

Publication: Clin Biochem, 2002, Vol. 35, Page 581-9

PubMed ID: 12498991 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the intra- and inter-individual variability and the effects of kidney disease on the protein profile of urine specimens. Twenty-four hour or morning urine from 49 healthy male patients, a patient with insulin-dependent diabetes mellitus and incipiens nephropathy, a patient with IgG monoclonal gammapathy and myelomatous kidney, a patient with idiopathic proximal tubular acidosis, and a patient with nephritic syndrome secondary to minimal change disease was collected into thymol-containing containers, aliquoted, frozen in liquid nitrogen, and then stored at -20˚C.  Additionally, specimens were obtained at 4-month intervals over the course of a year from three healthy individuals. Specimens were prepared by centrifugation at 3000 x g for 10 min, nucleic acids were precipitated from the supernatant with protamine sulfate at 4˚C, a second centrifugation at 10,000 x g for 20 min, precipitation of glycosaminoglycans with hydrochloric acid, dialysis, filtration, concentration, and finally lyophilization. Lyophilized urine was resuspended in a buffer containing SDS, DTE, urea, CHAPS, TRIS, and bromophenol blue before 2D PAGE. Proteins were digested with trypsin in the gel, extracted, dried, and redissolved in 5% formic acid before MALDI-TOF analysis. The protein analysis was not conducted for five specimens due to low protein abundance.

   Summary of Findings:

   When lactoferrin and trypsinogen were added to urine, 111% of lactoferrin and 92.3% of the trypsinogen were recovered and their mobility was not altered. When a single urine specimen was divided into three specimens and processed independently, variation was limited to less than 15% for the spots. Interestingly, the observed molecular weight and the calculated molecular weights and pI were different for several proteins, indicating the presence of posttranslational modification. Only slight variations in the protein pattern were observed among individuals and all specimens contained human serum albumin (HSA), α1-microglobulin (AMBP), Ig λ and κ light chains, complement regulatory protein 59 (CD 59), and C-terminal fragments of HSA (HSA-Cter). The protein excretion pattern only displayed small changes over the course of a year. The same proteins were present in morning spot urine and 24 h urine specimens collected from an individual on the same day and for most proteins the spot volume was within 20% of the other, but the morning spot urine specimen had a 66.6% higher concentration of one protein and 50% as much of another than the 24 h specimen. Finally, the urine protein profile differed among the diagnoses.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal
   • Diabetes Type 2
   • Other diagnoses
   • Diabetes Type 1

   Platform:

   Analyte	Technology Platform
   Peptide	MALDI-TOF MS
   Protein	1D/2D gels
   Protein	MALDI-TOF MS
   Peptide	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Diabetes mellitus and incipiens nephropathy IgG monoclonal gammapathy and myelomatous kidney Idiopathic proximal tubular acidosis Nephretic syndrome secondary to minimal change disease
   Biospecimen Acquisition	Method of fluid acquisition	Voided urine (24-h collection) Voided urine (spot collection)
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Multiple specimens analyzed
   Biospecimen Aliquots and Components	Biospecimen components	Lactoferrin Trypsinogen
   Biospecimen Acquisition	Time of biospecimen collection	0 months 4 months 8 months 12 months 24 h urine Morning urine

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