| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
Departments of1 Laboratory Medicine and2 Critical Care Medicine, Warren Magnuson Clinical Center, NIH, Bethesda, MD
aaddress correspondence to this author at: Department of Laboratory Medicine, NIH, Bldg 10, Room 2C-407, Bethesda, MD 20892-1508; fax 301-402-1885, e-mail ghortin{at}mail.cc.nih.gov
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 
1-microglobulin, albumin, IgG, and 2-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 for protein concentration—centrifugal ultrafiltration, acetone precipitation, acid precipitation, dye precipitation, ultracentrifugation, and lyophilization—generally have drawbacks of poor peptide recovery, poorly soluble pellets, or failure to remove salts (6). We examined solid-phase extraction of urinary peptides, using a polymeric sorbent with a pore size that should exclude albumin and other proteins of similar or greater size.
Urine specimens were processed in 6-mL cartridges containing 500 mg of StrataTM-X polymeric sorbent (Phenomenex) on a vacuum manifold. Pore size of the sorbent was specified by the manufacturer to be 91 Å, yielding a predicted size exclusion limit of 
20 000 Da. Cartridges were primed with 4 mL of methanol followed by 4 mL of 5 g/L acetic acid before addition of urine specimens, which had been acidified with acetic acid during collection to a pH of 4–5. After extraction of urine, cartridges were washed with 8 mL of 5 g/L acetic acid, and peptides were eluted with 3-mL steps of increasing acetonitrile concentration or with 600 mL/L acetonitrile–5 g/L acetic acid. Measurements of total protein and albumin to determine the amounts of proteins eluted from the cartridges were performed by standard methods (pyrogallol red and immunoturbidimetry, respectively) on a LX-20 analyzer (Beckman-Coulter). C-Peptide was measured by competitive immunoassay with an Immulite 2000 (Diagnostics Products Corp.). Eluates from extraction cartridges were analyzed either after evaporation under nitrogen to 2 mL or directly.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry was performed with an Ultraflex TOF mass spectrometer (Bruker Daltonics) in a linear positive-ion mode. Specimens were applied manually to 384-position target plates as 1-µL aliquots between layers of matrix applied as 1-µL aliquots of 10 g/L sinapinic acid in 750 mL/L acetonitrile–250 mL/L water containing 10 g/L acetic acid. Data were summed for 300 laser pulses collected from 10 positions. Measurements of mass/charge (m/z) were by external calibration. Calibrators and sinapinic acid were purchased from Bruker Daltonics.
Solid-phase extraction of highly proteinuric urine (Table 1
) served as good example of the size selectivity and binding capacity of the extraction cartridge. We loaded 10-mL aliquots of urine successively on a single cartridge and analyzed the eluates. The albumin concentrations of flow throughs were approximately the same as the initial concentrations, indicating very low extraction of albumin. Elution of C-peptide, which has a mass of 3000 Da, was monitored with a quantitative immunoassay. Flow through concentrations of C-peptide were 1% of the initial concentration even after loading of 50 mL of the proteinuric specimen, providing evidence that the capacity of the cartridge was not reached even with this large specimen load. Stepwise elution of components bound to the column by increasing concentrations of acetonitrile yielded the largest amounts of C-peptide at 300 mL/L acetonitrile. The C-peptide concentration in the 300 mL/L acetonitrile eluate was 10-fold higher than in the initial specimen, whereas the albumin concentration was 2% of the original. The amount of C-peptide was 78% of the original for the two eluates at 300 and 400 mL/L acetonitrile compared with only 0.3% for albumin. Analysis of the C-peptide concentrations obtained for seven different 50-mL urine specimens (ranging from normal to high protein) by single-step elution with 600 mL/L acetonitrile gave mean (SD) values that were 80 (20)% of the amounts loaded on the cartridges. In analyses of 15 aliquots of a single specimen containing 1010 mg/L albumin and 32.4 µg/L C-peptide, the amounts of albumin and C-peptide eluted by acetonitrile were 2.4 (0.3)% and 106 (7)%, respectively, of the amounts loaded on the cartridges.
|
The low binding capacity of the cartridge for albumin (1 mg) probably results from steric exclusion of albumin from pores of the polymeric solid-phase adsorbent. Albumin, with a molecular mass of 67 000 Da, is well above the expected size exclusion limit for pores of the adsorbent. Therefore, low-capacity binding of albumin occurs only on the external surface of the adsorbent, and albumin binding saturates without affecting binding of small peptide components such as C-peptide; the internal surface of pores represents the major surface area for capture of small peptide components.
Extraction of urine greatly improved the ability to detect peptide components by MALDI (Fig. 1
). In the m/z range 1250–5000, few components were apparent in unprocessed urine, and signals were very weak (Fig. 1
, top). Analysis of eluates at 400 mL/L acetonitrile showed several components for the proteinuric specimen analyzed in Table 1
(Fig. 1
, middle) and for a urine with a protein concentration (70 mg/L) within normal limits (Fig. 1
, bottom). The proteinuric specimen had a greater number of components and yielded stronger signals, indicating greater complexity and concentration of peptides. Components detected by mass spectrometry eluted at various acetonitrile concentrations. For the six fractions that were eluted with 100–600 mL/L acetonitrile, >100 different peaks were observed for the proteinuric specimen, and 25 peaks were detected in the specimen with a low protein concentration in the m/z range 1200–7000 (spectra not shown). It is likely that there are thousands of peptide components in urine and that the number observed is likely to depend on the sample preparation and the sensitivity and resolution of the method of analysis (7).
|
Results of the present study suggest that solid-phase extraction of urine with a polymeric solid phase provides a simple method for extraction of peptides for analysis by mass spectrometry. Peptides were eluted in a salt-free solution containing acetonitrile that might be concentrated further by evaporation. Elution of the greatest number of components at moderate concentrations of acetonitrile (300–400 mL/L) suggests that it should be possible to elute most components off the solid phase. Previously, solid-phase extraction has been applied as a tool for preparation of peptide mixtures, such as tryptic digests of proteins, for MALDI TOF mass spectrometry (12)(13)(14). A specialized variation of solid-phase extraction that has been termed surface-enhanced laser desorption/ionization (SELDI) directly extracts peptides onto the target surface for mass spectrometry (15). Typically, solid-phase extraction of peptides has involved use of octadecylsilica as the adsorbent, although some reports note that recovery of hydrophilic peptides can be increased by use of graphite particles or a mixed bed of graphite and octadecylsilica (12)(13)(14). Often, sample preparation for MALDI TOF mass spectrometry has been performed in pipette tips packed with tiny amounts of adsorbent. This format provides adequate specimen for MALDI TOF mass spectrometry but not enough for other, traditional clinical laboratory techniques. In addition, octadecylsilica or graphite adsorbents are likely to lack the size selectivity observed in the present experiment because of the larger pore sizes of common adsorbents.
Polymeric adsorbents have potential advantages with respect to capacity and suitability for large volumes of aqueous specimen (12). High capacity is a desirable characteristic in that it allows concentration of larger volumes of the relatively dilute peptide solutions in urine. The solid-phase extraction procedure described here permits the processing of substantial volumes of specimen and yields eluates of sufficient volume to combine mass spectrometric analysis with traditional clinical laboratory assays that can assess specimen recovery or measure components below the detection limits of mass spectrometry. Size-selective extraction of peptides may be of greatest value for fluids such as proteinuric urine or plasma, which contain high concentrations of albumin or other large proteins. For many diagnostic purposes, it may be useful to combine quantitative assays of specific components such as C-peptide with the high-resolution qualitative analysis provided by MALDI TOF mass spectrometry. The extraction technique here, which is directed at the concentration of peptide and small protein components (<20 000 Da), is complementary to the ultrafiltration techniques that are commonly used in clinical laboratories to concentrate proteins >10 000 Da in urine specimens. The size selectivity of the present extraction technique provides high enrichment of small peptide components such as C-peptide vs a protein such as albumin with a molecular mass of 67 000 Da. In the present study, we did not quantify the amounts of any proteins near the predicted size exclusion limit of 20 000 Da. Retention of such components is likely to be affected by their molecular shape and to be intermediate in efficiency between small peptides such as C-peptide and proteins the size of albumin.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  D. Sviridov, S. K. Drake, and G. L. Hortin Reactivity of Urinary Albumin (Microalbumin) Assays with Fragmented or Modified Albumin Clin. Chem., January 1, 2008; 54(1): 61 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Fiedler, S. Baumann, A. Leichtle, A. Oltmann, J. Kase, J. Thiery, and U. Ceglarek Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Clin. Chem., March 1, 2007; 53(3): 421 - 428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Hortin The MALDI-TOF Mass Spectrometric View of the Plasma Proteome and Peptidome Clin. Chem., July 1, 2006; 52(7): 1223 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Hortin and B. Meilinger Cross-Reactivity of Amino Acids and Other Compounds in the Biuret Reaction: Interference with Urinary Peptide Measurements Clin. Chem., August 1, 2005; 51(8): 1411 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Drake, R. A.R. Bowen, A. T. Remaley, and G. L. Hortin Potential Interferences from Blood Collection Tubes in Mass Spectrometric Analyses of Serum Polypeptides Clin. Chem., December 1, 2004; 50(12): 2398 - 2401. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
