NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

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

This paper investigated intra and inter-individual variability and the effects of kidney disease on the protein profile of urine specimens.

Conclusion of Paper

All urine specimens from healthy individuals 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), but slight inter-individual variations were observed and the protein profile differed from individuals with kidney disease. Minimal intra-individual variability of the protein profile was observed over the course of a year and the same proteins were present in morning spot urine and 24 h urine with changes in abundance only noted for two proteins.

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
    Preservative Types
    • Other Preservative
    Diagnoses:
    • Normal
    • Diabetes Type 2
    • Other diagnoses
    • Diabetes Type 1
    Platform:
    AnalyteTechnology Platform
    Peptide MALDI-TOF MS
    Protein 1D/2D gels
    Protein MALDI-TOF MS
    Peptide 1D/2D gels
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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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