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Proteomics and Protein Markers |
Departments of1 Laboratory Medicine and 2 Critical Care Medicine, Warren Magnuson Clinical Center, National Institutes of Health, Bethesda, MD.
aAddress correspondence to this author at: Department of Laboratory Medicine, National Institutes of Health, Building 10, Room 2C-407, Bethesda, MD 20892-1508. Fax 301-402-1885; e-mail ghortin{at}mail.cc.nih.gov.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We evaluated separation of purified proteins and urinary components by size-exclusion HPLC using a Zorbax Bio Series GF-250 column eluted with phosphate-buffered saline. Urinary components eluting in the "albumin" peak were analyzed by mass spectrometry and reversed-phase HPLC.
Results: Several proteins, such as transferrin, 
1-proteinase inhibitor, 1-acid glycoprotein, and 2-HS glycoprotein, analyzed as purified components, were not resolved from albumin by size-exclusion HPLC. Peaks for other proteins, such as IgG and urinary components identified as dimers of 1-microglobulin and immunoglobulin light chains, overlapped with the albumin peak. Profiles of urine specimens showed variable amounts of components overlapping with albumin. Furthermore, the albumin peak obtained by size-exclusion HPLC was found by mass spectrometry and reversed-phase HPLC to contain multiple components in addition to albumin.
Conclusions: Size-exclusion HPLC does not resolve albumin from several other proteins in urine. The albumin peak resolved by this technique, although predominantly composed of albumin, contains several coeluting globulins that would contribute to overestimation of albumin concentration by size-exclusion HPLC.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent development in the measurement of urinary albumin has been the application of size-exclusion HPLC in a commercially available method (Accumin®; AusAm Biotechnologies) to quantify urinary albumin (6)(7)(8)(9)(10)(11). This assay has yielded consistently higher measured albumin values for urine specimens than have immunoassay techniques (6)(7)(8)(9)(10)(11). It has been hypothesized that the higher values with the HPLC assay may result from modified forms of albumin of the same size as albumin but with reduced immunoreactivity. It has been proposed that measurement of albumin by the HPLC method might offer diagnostic advantages over albumin measured by immunoassays (6)(7)(8)(9).
In the present study, we examined the resolution of major urinary proteins by size-exclusion HPLC. Size-exclusion chromatography, even in its highest resolution forms, generally has difficulty separating proteins of similar relative molecular mass (Mr) (12). Separations by this technique relate to differences in hydrodynamic radius, which for globular molecules increases proportionally only with the cube root of molecule weight (13)(14). The radii of globular proteins such as human serum albumin and transferrin with relative molecular masses of 
67 000 and 77 000 are therefore nearly the same, 35 Å vs 36 Å (13). Molecular shape and charge exert secondary effects on separations by size-exclusion chromatography (12)(13)(14).
Although albumin is the most abundant plasma protein, there are many other plasma proteins of approximately the same molecular size, such as transferrin, 1-proteinase inhibitor, hemopexin, 1-acid glycoprotein, 1-antichymotrypsin, Gc-globulin, 2-HS glycoprotein, and transthyretin, that are major urinary components (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Fundamental principles suggest that size-exclusion HPLC will not resolve albumin from other urinary proteins of similar molecular size. We therefore analyzed albumin and other urinary components by a size-exclusion HPLC method developed in our laboratory to determine whether other major urinary proteins coeluted with albumin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analytical size-exclusion chromatography was not performed with the commercially available Accumin (AusAm Biotechnologies), but used the same type of column as in published HPLC analyses of urine components (6)(7)(8)(9)(10)(11), a Zorbax Bio Series GF-250 column with dimensions of 9.4 (i.d.) x 250 mm (Agilent Technologies). Analyses used phosphate-buffered saline (9 g/L NaCl, 0.775 g/L Na2HPO4, 0.165 g/L KH2PO4, pH 7.4) at a flow rate of 1 mL/min and were performed with a system equipped with a Model 717 autosampler, Model 626 pump, and Model 996 photodiode array detector (Waters Corp.). The injected sample volume was 25 µL. Reversed-phase HPLC used the same HPLC system with a 3.9 (i.d.) x 150 mm Delta Pak C18 column (5-µm particles with a 300Å pore size) eluted with a linear gradient from 30% to 60% acetonitrile over 30 min (buffer A, 1 mL/L trifluoroacetic acid in water; buffer B, 0.8 mL/L trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min. The injected sample volume was 100 µL, and the samples were the central fractions of albumin peaks from size-exclusion HPLC analyses of urine specimens.
Preparative size-exclusion chromatography of urine proteins, with a specimen injection volume of 2 mL, on a Pharmacia FPLC system was performed with a 1.6 (i.d.) x 60 cm Sephacryl S-200 column (Amersham Biosciences) eluted with phosphate-buffered saline.
Total protein and urinary albumin were determined by standard clinical methods on a Beckman-Coulter LX-20 analyzer. Quantitative analysis of urine by HPLC was performed with calibrators prepared from pure albumin. Calibrators yielded a linear calibration curve of peak area vs concentration, and calibrator values were matched with immunoassay values. Agarose gel electrophoresis was performed on a Paragon system (Beckman-Coulter). 1-Microglobulin was quantified on a BN-II nephelometer (Dade Behring).
Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze components in the albumin peak from size-exclusion HPLC. Aliquots (10 µL) of each specimen were diluted with 90 µL of binding buffer (100 mmol/L Tris, pH 9.0, for Q10 anion-exchange target surfaces, or 100 mL/L acetonitrile–1 mL/L trifluoroacetic acid for H50 hydrophobic target surfaces; Ciphergen Biosystems) and incubated with shaking for 2 h in a 96-well bioprocessor. All samples were run in duplicate, and all liquid handling steps were performed with a Biomek2000 automated workstation (Beckman Coulter). After incubation, target surfaces were washed 3 times with binding buffer and twice with deionized water, and were air-dried for 15 min. Two 0.5-µL application of saturated sinapinic acid in 500 mL/L acetonitrile–5 mL/L trifluoroacetic acid were made to each target surface. Mass spectra were summed for 130 laser shots with a Ciphergen Protein Biological System II externally calibrated with proteins of the following relative molecular masses: hirudin BKHV, Mr 7032; equine cytochrome c, Mr 12 360; equine myoglobin, Mr 16 952; bovine carbonic anhydrase, Mr 29 024; yeast enolase, Mr 46 671; and bovine albumin, Mr 66 433.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), transferrin (Fig. 1B
), 1-acid glycoprotein (Fig. 1C
), and 1-proteinase inhibitor (also known as 1-antitrypsin; Fig. 1D
). Each of these proteins yielded elution profiles with major peaks at approximately the same time as albumin and minor earlier peaks, possibly corresponding to small amounts of protein dimers. The elution times of the major components for these proteins were all within 0.1 min of each other. We analyzed mixtures of 2 protein components to determine whether the separate peaks could be resolved. Chromatography of approximately equal amounts of protein should provide the optimal opportunity to resolve peaks. However, as shown for the mixture of albumin and transferrin (Fig. 1E
), the evaluated method failed to resolve mixtures of any of the above proteins with albumin into 2 distinct peaks. No separation of components could be achieved because peak widths were greater than the difference in elution time between components. Chromatograms appeared to have peak widths similar to published profiles obtained with the method for urine albumin measurement (6). Varying the concentration of injected proteins over a range from 0.01 to 1 g/L did not change peak widths, so that protein overloading did not account for the inability to resolve these components (not shown).
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Repetitive analysis of these proteins showed that within-run elution times with the size-exclusion HPLC method were highly reproducible (Table 1
). The very small differences in the elution times, <0.1 min, between these 4 components were not sufficient to achieve separations of protein mixtures, however. Peaks widths measured at half peak height were 0.2–0.3 min, substantially greater than the differences in elution times. Although these proteins differ somewhat in relative molecular mass, from 40 000 to 80 000, separations by size-exclusion chromatography are dependent on the shape and hydrodynamic radius of proteins rather than directly on their molecular mass. High oligosaccharide content for a protein such as 1-acid glycoprotein may confer a greater hydrodynamic radius per unit of mass. Separations on size-exclusion columns may also be influenced by the charge of molecules as a result of charge-exclusion or ion-exchange effects (12)(13). We evaluated the influence of these effects by changing the ionic strength or pH of the eluent. Decreasing the ionic strength by half or adjusting the pH of the elution buffer from 7.4 to 7.0 had only a small effect on the elution times of these components; therefore, modest variations in the buffer composition did not allow separation of albumin from the globulins.
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We analyzed several other purified protein components to establish the relationship between molecular size and elution time (Table 2
). Some additional proteins, such as Gc-globulin and 2-HS glycoprotein, had elution times that would lead to coelution with albumin. Haptoglobin type 1-1 and IgG eluted slightly earlier, whereas prealbumin and urine fractions enriched in 1-microglobulin and immunoglobulin light chain had major peaks eluting slightly later than albumin. The latter 2 components eluted much closer to albumin than expected, possibly because of dimerization, which has been noted previously to occur for these proteins (26)(27)(28)(29). These proteins were obtained by preparative chromatography of normoproteinuric urine and urine from a patient with myeloma, respectively, and were characterized by immunoassays and agarose gel electrophoresis as well as size-exclusion chromatography.
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Purified Tamm–Horsfall protein eluted predominantly in the excluded volume, and the peak had an extended tail, but there was no significant overlap with albumin (analysis not shown). This protein, often the most abundant urinary protein, is known to aggregate at physiologic ionic strength and to dissociate at low ionic strength (25)(30). When the ionic strength of the elution buffer was halved, analysis of the Tamm–Horsfall protein yielded a pattern suggesting occurrence of monomers, dimers, and trimers of a protein with a relative molecular mass of 90 000 (data not shown), which is the size of the Tamm–Horsfall protein subunit (30).
The elution times of proteins had an overall linear relationship vs log(Mr), but there was significant variance of individual components from this relationship (Fig. 2
). In particular, IgG and 1-antichymotrypsin diverged from the usual relationship between size and elution time and eluted at later times than expected (identified as points 1 and 2 in Fig. 2
). Elution of these proteins appeared to be delayed because of adsorption on the column. Peaks for these proteins showed extensive tailing. Doubling the ionic strength of the elution buffer moved their elution times toward expected values (Table 2
) and decreased peak tailing. Ionic strength–dependent adsorption of selected proteins during size-exclusion chromatography is a previously described problem (12)(13).
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The delayed elution and tailing of IgG (Fig. 3A
) caused it to overlap substantially with albumin (Fig. 3B
) as shown by chromatography of a specimen containing an 1:1 mixture of these proteins (Fig. 3C
). The same albumin concentration was in the mixture and in the specimen with albumin alone, which allowed evaluation of the ability to accurately integrate the albumin peak in the presence of an overlapping protein. In this case, the severe tailing of the IgG peak underlying the albumin peak (shown as a dashed line in Fig. 3C
) caused overestimation of the peak area of the albumin peak by a mean of 66% for triplicate measurements, even when the extended tail trailing the albumin peak was eliminated by ending the albumin peak at 10.2 min. Peak areas were highly reproducible with an SD <1% of the total peak areas of the albumin peak in either a mixture or pure solution. Although the peak areas for albumin were reproducible, this example illustrates how integration of the albumin peak can be inaccurate in the presence of a nonsymmetric overlapping peak.
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Analysis of a set of 8 urine specimens showed that, in some cases, it was difficult to identify the limits of the albumin peak for integration. The albumin peak often was asymmetric with apparent shoulders before or after the peak, as shown in Fig. 4A
. There is some operator dependence if limits are set manually, and automated integration would depend on the settings used for establishing peak limits. Some potential variations of the baseline are shown in Fig. 4A
as dashed lines a, b, and c. An example of a more symmetric, albeit wider than for calibrators, peak for albumin is shown in Fig. 4B
. It is not clear in published reports whether the methods used for albumin analysis integrate peaks on a trough-to-trough basis (dashed line a in Fig. 4B
) or integrate to the baseline, which would lead to substantial overestimation of albumin. Comparison of albumin measurements by HPLC vs immunoassay (Fig. 4C
) yielded close agreement with immunoassay values when peak limits were assigned manually, such as line a in Fig. 4B
. Albumin values by HPLC were 2-fold higher than immunoassay if integration was to the baseline in the absence of protein. From these analyses, we conclude that the method of peak integration is an important variable and that in some cases it is difficult to define the limits of the albumin peak for integration.
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From the results shown in Fig. 1
and Tables 1
and 2
, it was clear that the size-exclusion HPLC method could not resolve several proteins from albumin as purified components. However, there remained the issue of whether these proteins would coelute with albumin in significant amounts in actual urine specimens. We performed a detailed analysis of components in the albumin peak of urine specimens separated by size-exclusion HPLC for 2 urine specimens that were dipstick-negative for protein. These specimens were concentrated by ultrafiltration and analyzed by size-exclusion HPLC. The first specimen had an albumin concentration (33 mg/L) in the microalbuminuric range and a total protein of 130 mg/L. Analysis by size-exclusion HPLC showed a primary peak in the expected position for albumin and smaller overlapping peaks preceding and following albumin (Fig. 5A
). Further analysis of a fraction corresponding to the central portion of the albumin peak (indicated by shading in Fig. 5A
) from the size-exclusion HPLC separation showed that there were several other components in addition to albumin in this fraction. Reversed-phase HPLC revealed that approximately two thirds of the protein absorbance eluted in the position of albumin near 14.8 min (Fig. 5B
). Other components eluted at the positions of calibrators for transferrin and 1-acid glycoprotein (both of which eluted near 12.6 min) and 1-proteinase inhibitor at 23.9 min. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry performed with a hydrophobic target surface detected a diverse range of products in addition to the major peak for albumin, which in this analysis was the peak at m/z 66 671 (Fig. 5C
). Other peaks, including a peak corresponding to the expected m/z for transferrin, were revealed by analysis with an anion-exchange target surface (Fig. 5D
).
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Similar analysis of a second urine specimen with an albumin concentration within the reference limits by immunoassay (5 mg/L) and a total protein of 130 mg/L showed a much smaller proportion of the total protein eluting in the position of albumin (Fig. 6A
). Analysis the albumin peak by reversed-phase HPLC and mass spectrometry showed the presence of multiple other components in addition to albumin (Fig. 6
, B, C, and D). When we analyzed the albumin peak by reversed-phase HPLC, albumin appeared to comprise approximately two thirds of the total protein absorbance at 214 nm, and peaks were observed in the positions expected for transferrin/1-acid glycoprotein and for 1-proteinase inhibitor. Mass spectrometry gave peaks corresponding to the m/z of protein calibrators for 1-acid glycoprotein (34 000–39 000), 1-proteinase inhibitor (50 000), and transferrin (78 000), and there were multiple other unidentified peaks (Fig. 6
, C and D) for proteins captured on hydrophobic and anion-exchange target surfaces, respectively.
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In the 2 analyses of urine specimens by size-exclusion HPLC shown (Figs. 5A
and 6A
), the "albumin" peak (shown above to consist of a mixture of components such as albumin, transferrin, 1-acid glycoprotein, 1-proteinase inhibitor, and other unidentified components) was not completely resolved from other peaks. Other overlapping components formed shoulders preceding and following the albumin peak. In addition to the contribution of components that actually coeluted with albumin in this method, additional overlapping components may hinder the ability to accurately delineate and integrate the peak for albumin as shown for the example of IgG above.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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70% of the albumin peak from size-exclusion HPLC. Although we used a different HPLC method, we cannot support the previous report that the albumin peak from size-exclusion HPLC contained undetectable amounts of several globulins (6). We conclude that size-exclusion HPLC will systematically overestimate the concentration of albumin in urine and that the albumin peak obtained by this method actually represents the combined contribution of albumin plus globulins with sizes similar to that of albumin.
The amounts of albumin-sized globulins coeluting with albumin is expected to be 20%–30% based on several considerations. The aggregate concentration in plasma of globulins with a molecular size similar to albumin, proteins such as transferrin, 1-acid glycoprotein, 1-proteinase inhibitor, 1-antichymotrypsin, 2-HS glycoprotein, hemopexin, Gc-globulin, and many others (31), in healthy individuals is 25% that of albumin; this aggregate concentration is even higher during acute-phase reactions (32). Because the passage of plasma proteins through the glomerular barrier into urine is primarily a size-dependent process with a size-exclusion limit approximately the size of albumin (33), it is not surprising that all of the albumin-sized plasma components are also found in urine (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(33)(34)(35)(36)(37)(38)(39). There is considerable variation in the reported proportions of individual globulins relative to albumin in urine (Table 3
). However, the aggregate concentration of albumin-sized globulins in urine appears to be 20% that of albumin, with significant variation in different physiologic states. During acute-phase responses, for example, the urinary concentration of 1-acid glycoprotein reaches 80% that of albumin (24).
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A second problem with the size-exclusion HPLC analysis is that several urinary components, although not exactly coeluting with albumin, overlap considerably. As listed in Table 3
, the total concentrations of these components, such as IgG, immunoglobulin light chains, and 1-microglobulin, could approach or exceed the albumin concentration (21)(24)(37)(40)(41). By virtue of its much larger size than albumin, IgG would be expected to be well resolved from albumin; however, under the standard conditions of size-exclusion HPLC analysis, it eluted much later than expected, probably because of adsorption on the column. As a result, it overlaps with the albumin peak. Immunoglobulin light chains and 1-microglobulin also would be expected to elute well after albumin, but we observed that some of their urinary forms, probably dimers, as reported by others (26)(27)(28)(29), overlapped with albumin. The overlap of other components with albumin could lead to some guesswork in deciding where the true baseline for the albumin peak lies. This may interfere with the accuracy of albumin measurements by this method. It is difficult to predict the magnitude of this inaccuracy, as it will depend on the settings used for integration as well as the relative concentrations of overlapping components. Difficulties with accurate integration of the albumin peak are expected to be most severe when albumin concentrations are low and the relative proportions of overlapping components are high, such as for the example shown in Fig. 6A
. It is not clear how any meaningful quantification of albumin could be performed for analyses such as this with no clear resolution of albumin from overlapping components. Depending how integration of the albumin peak was performed, we found for a small set of concentrated urine specimens that the HPLC values ranged from approximately equivalent to approximately twice as high as those obtained by immunoassay. With this variance and uncertainty in integration, it is difficult to assess the quantitative contribution of coeluting globulins to the albumin peak.
Previous reports have suggested that one source of higher albumin measurements by HPLC vs immunoassay methods was the presence of nonimmunoreactive albumin (6)(7)(8)(9)(10)(11). We do not rule out the presence of such a component in some specimens, although our results suggest that contributions of globulins explain at least part of the higher values obtained by HPLC vs immunoassay. The authors of other studies have reported the presence of significant amounts of albumin fragments as detected by 2-dimensional electrophoresis (16), and the possibility of some modification of urinary albumin has been considered for many years because of slightly altered electrophoretic mobility (18)(36). Further investigation is indicated to determine whether there are significant amount of "nonimmunoreactive albumin", as suggested previously, or whether the increased amounts of albumin measured by HPLC are mainly attributable to other components eluting at or near the position of albumin. In one recent large-scale trial, the systematic bias of HPLC vs immunoassay, yielding values averaging 26% higher in the microalbuminuric range (20–200 mg/L albumin) for 280 persons, can be accounted for nearly completely by the expected contributions of glycoproteins coeluting with albumin in the HPLC method (10). Contributions of nonimmunoreactive albumin to values measured by HPLC therefore must have been minimal within this range of values. In that trial, biases of HPLC vs immunoassay were higher at low albumin values and lower at high albumin values. At low albumin concentrations, the proportions of components overlapping with albumin will be increased, and this may lead to greater overestimation of values by HPLC in this range.
In conclusion, we believe that size-exclusion HPLC, previously described as a method for measuring urinary albumin (6)(7)(8)(9)(10)(11), does not provide specific measurement of urine albumin. Instead, it provides a measure of the sum total of albumin and several globulins. In general, it has been observed that the urinary excretion of globulins such as transferrin and 1-acid glycoprotein correlates with urinary excretion of albumin (22)(23). Consequently, it is not clear what diagnostic advantage would accrue from measuring the sum total of albumin plus several globulins. Because the HPLC method for albumin analysis provides results that are not equivalent to those obtained by immunoassays and probably measures different molecular entities, extensive clinical trials would be required to establish appropriate reference intervals and decision levels for clinical interpretation. Studies of reference intervals for albumin values from the HPLC assay indicate that ranges are 2-fold higher than for immunoassays (11); therefore, efforts to apply the same diagnostic cutoff values that have been established for immunoassay methods to albumin measurements by size-exclusion HPLC, as in some recent studies (6)(7)(8)(9), are inappropriate. From the standpoint of interest in proteomic analysis of urine (15)(16)(17), size-exclusion chromatography appears to be a useful technique for producing a low-resolution size fractionation of urinary proteins. This fractionation appears to be a useful preparative step and precursor to other analytical techniques, some of which were applied to specific chromatographic fractions in this study.
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Acknowledgments |
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References |
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[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
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) correspond to IgG and 
