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.
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.
- Other Preservative
- Diabetes Type 2
- Other diagnoses
- Diabetes Type 1
Analyte Technology Platform Peptide MALDI-TOF MS Protein 1D/2D gels Protein MALDI-TOF MS Peptide 1D/2D gels
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
Biospecimen Acquisition Time of biospecimen collection 0 months
24 h urine