Article pubs.acs.org/jpr
Multiplexed LC−MS/MS Assay for Urine Albumin Ashley Beasley-Green,* Nijah M. Burris, David M. Bunk, and Karen W. Phinney Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8390, United States S Supporting Information *
ABSTRACT: Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. Although intermethod differences and analyte heterogeneity have been reported for urine albumin measurements, accuracy assessments of the available methods have been hindered by the lack of a reference system, including reference measurement procedures and reference materials, for this clinical analyte. To address the need for a reference measurement system for urine albumin, we have developed a candidate reference measurement procedure that utilizes isotope dilution−mass spectrometry (ID−MS) and multiple reaction monitoring (MRM) to quantify full-length urine albumin in a targeted mass spectrometric-based approach. The reference measurement procedure incorporates an isotopically labeled (15N) full-length recombinant human serum albumin (15N-rHSA) material as the internal standard, which permits the absolute quantitation of albumin in urine. A total of 11 peptides with two transitions per peptide were selected from the tryptic digestion of human serum albumin on the basis of retention time reproducibility, peak intensity, and the degree of HSA sequence coverage. In addition to method validation, the generated calibration curves were used to determine the albumin content in pooled human urine samples to access the accuracy of the MSbased urine albumin quantitation method. KEYWORDS: Urine albumin, reference measurement procedure, absolute quantitation, multiple reaction monitoring (MRM), isotope dilution−mass spectrometry (ID−MS)
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INTRODUCTION Urine albumin is a major biomarker for early diagnosis, disease management, and prognosis of renal disease and is critical for clinical decisions associated with renal therapy. Therefore, precise measurement of urine albumin plays an important role in the early detection of renal dysfunction, evaluation of treatment efficacy, and reduction in the risk of kidney failure and cardiovascular disease.1 Urine is produced by the kidney via the process of ultrafiltration and is primarily used to eliminate soluble waste products, such as electrolytes, nitrogenous compounds, and hormones, from plasma.2 Most of the low molecular weight proteins and highly abundant plasma proteins (i.e., albumin and immunoglobulins) filtered from plasma to the ultrafiltrate are reabsorbed in the proximal renal tubules.2 Normal excretion of protein in urine is less than 150 mg/L per 24 h, and urine is composed of approximately 10 mg/L of albumin.3 Due to the abundance of albumin in human plasma and its subsequent presence in urine, albumin has become the key protein used in the assessment of urinary excretion of plasma proteins. Native mature albumin is a globular protein produced in the liver that contains 585 residues (removal of signal sequence aa 1−24) arranged into three distinct domains and functions as a transport protein in plasma and a regulator of plasma oncotic pressure.4 Normal urine albumin levels range This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society
from 0 to 30 mg/L (normoalbuminuria); however, increased excretion of albumin in urine is divided into two groups: microalbuminuria (30−300 mg/L) and macroalbuminuria (>300 mg/L).5 To selectively measure albumin in urine, current clinical methodologies utilize affinity-based techniques with monoclonal and polyclonal antibodies; the clinical immunoassays for urine albumin include ELISAs, immunoturbidity assays, and radioimmunoassays.6,7 In addition to albumin-specific measurements, the albumin−creatinine ratio (ACR) is a widely used measurement to assess urine albumin content that corrects for variability in urinary protein composition.8 Creatinine is a product of creatine phosphate metabolism in muscle and is filtered out of the plasma via urine at a fairly constant rate.9 In the ACR measurement, creatinine is used to normalize urinary protein composition based on the constant excretion of creatinine in a given individual. Although albumin-specific assays are routinely used in clinical laboratories to assess patient samples, there are distinct measurement challenges for the current affinity-based methods. Several studies have identified albumin fragments, N- and Cterminal truncation products and glycated species, in addition Received: March 6, 2014 Published: July 24, 2014 3930
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selectively targets full-length albumin for the accurate and precise quantitation of urine albumin.
to the native full-length albumin in the urine of both healthy and diseased individuals.10−18 The presence of modified or fragmented forms of albumin in urine can alter the antibody recognition site for routinely used immunochemical assays and thus potentially bias the total albumin measurement. Variations of the urine albumin species also affect the analytical precision and intra- and interlaboratory measurement comparability of commonly used affinity-based assays.11 Due to the clinical importance of urine albumin measurement, the National Institute of Standards and Technology (NIST) has been assisting the National Kidney Disease Education Program (NKDEP) and the Working Group for the Standardization of Albumin Assays in Urine (WG-SAU) of the International Federation of Clinical Chemistry (IFCC) in the development of a reference measurement system for urine albumin.11 In support of this effort, we have developed a highly selective and sensitive mass spectrometry-based candidate reference measurement procedure to quantify urine albumin and to certify higher-order calibrators and secondary reference materials. Our approach incorporates isotope dilution−mass spectrometry (ID−MS) using liquid chromatography coupled to a triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) scan mode to measure proteolytic albumin fragments in urine.19−22 To target full-length albumin and to reduce bias introduced by sample preparation and enzymatic fragmentation, we utilize an isotopically labeled (15N) full-length recombinant human serum albumin (15NrHSA) as the internal standard. The full-length internal standard is chemically equivalent to the native analyte; therefore, the proteolytic products and retention times are identical.23 The availability of an 15N-rHSA internal standard enables the quantitation of multiple regions of the albumin sequence to reduce the influence of albumin heterogeneity and to improve the urine albumin measurement quality. Our approach builds on the liquid chromatography−tandem mass spectrometry (LC−MS/MS) method of Lieske et al.;7 however, our method incorporates the multiplexed measurement of 11 tryptic peptides from albumin rather than one to three peptides in the published method. Multiplexing has the potential to increase the precision of measurement, which is essential in the certification of higher-order calibrators and reference materials. This potential increase in precision will come without any additional sample preparation, as a single tryptic digest of albumin (and the 15N-rHSA internal standard) releases all peptides together. Additionally, mass spectrometers are capable of highly multiplexed measurements without a negative impact on measurement precision of individual peptides or increasing analysis time. As such, measurement of a higher number of tryptic peptides does not change the resources or the time needed for urine albumin measurement. The measurement of a higher number of tryptic peptides can also potentially provide qualitative information on the heterogeneity of the urine albumin. With more peptides measured from the different domains of albumin, a comparison of the measurement results from each peptide may provide evidence of structural differences such as truncation or posttranslational modifications. The ability to evaluate molecular heterogeneity of albumin in urine samples will be very useful in the detection of disease-specific albumin species and also in future commutability assessment studies of certified reference materials. The objective of this study is to develop a MS-based multiplexed candidate reference measurement procedure that
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EXPERIMENTAL METHODS
Disclaimer
Certain commercial equipment, instruments, and materials are identified in this article to adequately specify the experimental procedure. Such identifications do not imply recommendations or endorsement by NIST nor do they imply that the equipment, instruments, or materials are necessarily the best available for the given purpose. Materials
MS-grade trypsin (Promega; Madison, WI), high-purity LC− MS grade water/formic acid and acetonitrile/formic acid (Honeywell Burdick and Jackson; Muskegon, MI), unlabeled recombinant human serum albumin and recombinant human serum albumin domains I, II, and III (Albumin Biosciences; Huntsville, AL), recombinant human serum albumin labeled with 15N was produced at NIST, and microalbumin control calibrators (Randox Laboratories-US; Kearneysville, WV) were purchased as indicated. In-Solution Trypsin Digestion
Prior to in-solution digestion, the control albumin samples and urine samples were incubated at 95 °C for 10 min to denature the protein. The samples were reduced with 5 mmol/L dithiothreitol at 60 °C for 30 min followed by alkylation with 15 mmol/L iodoacetamide at room temperature for 30 min. An approximate 1:30 weight ratio of trypsin-to-total protein was used for digestion and incubated at 37 °C overnight (≈18 h). Following digestion, the pH of the sample was lowered with 50 mL/L formic acid in water and incubated for 45 min at 37 °C to quench the digestion reaction. The tryptic fragments were concentrated and resuspended in 1 mL/L formic acid in water. Ion Trap MS Analysis
General conditions for the LTQ-XL (Thermo Fisher; San Jose, CA) linear ion trap mass spectrometer were as follows: sheath gas flow rate of 45 (arbitrary units), auxiliary gas flow rate of 5 (arbitrary units), capillary temperature of 230 °C, normalized collision energy of 35.0%, activation q-value of 0.25, and activation time at 30 ms. The Top 4 data-dependent acquisition (DDA) experiment, where the top four most intense ions from the full MS (MS1) scan were targeted for MS/MS (MS2) analysis, was utilized in each study. The raw tandem MS data was processed with the Protein Discoverer software (version 1.4, Thermo Fisher), the UniProtKB/Swiss-Prot human serum albumin (P02768) fasta from http://uniprot.org created on April 1, 1990, was used to generate the fasta file included in the MS/MS search methods, and a cutoff correlation score of 1.5 was used for the final peptide identification list. A detailed description of the LC gradient, DDA parameters, and final protein/peptide identification list is outlined in the Supporting Information. Triple Quadrupole MS Analysis
Quantitative analysis was performed using an Agilent 6460 triple quadrupole mass spectrometer in the positive mode equipped with an Agilent 1290 Series LC system utilizing an Agilent Zorbax 300SB-C18 column (2.1 mm × 150 mm, 3.5 μm). The column temperature was maintained at 45 °C, and the peptides were loaded onto the column with a flow rate of 200 μL/min in 97% (v/v) mobile phase A (water with 1 mL/L 3931
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Figure 1. Amino acid sequence of human serum albumin with the MRM assay peptides highlighted in bold. The albumin domains (I, II, and III) are highlighted in shades of gray.
Table 1. Optimized Urine Albumin MRM Transitions time segment 1
2
3
4
5 6
peptide ID HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA HSA
11.1 11.3 7.1 7.2 1.1 1.2 4.1 4.2 5.2 5.4 2.1 2.2 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2 12.1 12.3 12.4
peptide
precursor ion
product ion
TYETTLEK TYETTLEK AEFAEVSK AEFAEVSK DLGEENFK DLGEENFK LCTVATLR LCTVATLR YLYEIAR YLYEIAR LVNEVTEFAK LVNEVTEFAK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK
492.75 492.75 440.42 440.42 476.23 476.23 467.25 467.25 464.25 464.25 575.31 575.31 480.79 480.79 500.81 500.81 637.64 637.64 507.30 507.30 682.37 682.37 682.37
720.38 265.12 201.09 680.36 723.33 229.12 660.40 274.12 651.35 277.16 937.46 694.38 276.13 685.44 488.31 587.38 961.46 244.17 189.12 712.40 970.52 712.44 899.98
IS precursor iona IS product iona 497.23 497.23 445.21 445.21 481.21 481.21 472.75 472.75 469.24 469.24 581.29 581.29 487.27 487.27 506.29 506.29 644.3 644.3 513.29 513.29 689.35 689.35 689.35
727.40 279.20 203.04 687.30 731.30 231.07 669.4 276.12 659.30 279.10 947.40 701.40 279.09 695.40 493.25 593.40 972.4 247.16 191.08 721.40 981.50 721.37 909.50
RT
CE
dwell
fragmentor
9.1 9.1 9.5 9.5 11.1 11.1 12.4 12.4 14.5 14.5 15.3 15.3 15.9 15.9 17.4 17.4 18 18 20.2 20.2 23.1 23.1 23.1
12 12 11 11 9 9 12 12 11 11 15 15 11 11 13 13 22 22 18 18 16 16 16
113 113 113 113 113 113 113 113 75 75 75 75 75 75 113 113 113 113 225 225 150 150 150
130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130 130
a15
N-labeled rHSA internal standard (IS).
nebulizer of 20 psi, sheath gas temperature of 300 °C, sheath gas flow of 6 L/min, capillary voltage of 4000 V, and a nozzle voltage of 1500 V.
formic acid) and 3% (v/v) mobile phase B (acetonitrile with 1 mL/L formic acid). The peptides were eluted from the hydrophobic stationary phase with a linear gradient, and the total gradient time was 40 min. Solvent B was held constant at 3% for 3 min and then ramped to 10% in 2 min and to 20% in 10 min, followed by an increase to 30% in 10 min. At 30 min, solvent B was increased to 97% and held constant for 2 min and ramped down to 3% in 3 min to re-equilibrate the reversedphase column. Clean and blank LC−MS/MS runs preceded each analysis set, which were run in order of increasing albumin content. General mass spectrometric conditions were as follows: gas temperature of 300 °C, gas flow of 7 L/min,
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RESULTS AND DISCUSSION
MRM Transition Selection and Optimization
Peptides function as surrogates for proteins in targeted proteomic measurements to indirectly determine full-length protein concentrations; therefore, peptide specificity is critical for accurate quantitative protein measurements. To evaluate full-length urine albumin content, we utilized both ion trap and 3932
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triple quadrupole MS data to select optimal peptides that span the HSA amino acid sequence (Figure 1). From ion trap MS analysis of tryptic digests of both unlabeled and 15N-labeled recombinant albumin (rHSA), we identified 82 unlabeled (89.98% protein sequence coverage) and 78 labeled (84.56% protein sequence coverage) peptides, and the presence of each peptide was observed in three technical runs (Supporting Information Table S4). The unlabeled material was utilized to develop the multiplexed MRM assay, and a total of 33 peptides were selected for further investigations. The MRM peak intensity was used to determine the optimal precursor ion charge state for each peptide, and the MRM peak area was evaluated to determine the optimal product ion transitions for the multiplexed assay (Supporting Information Tables S5 and S6). Additionally, to enhance assay sensitivity, the MRM peak intensity was utilized to assess the optimal collision energy (CE), dwell time, and fragmentor voltage for each transition (Supporting Information Tables S7−S9). A total of 11 peptides with two transitions (product ions) per peptide were selected for the multiplexed urine albumin quantitation assay on the basis of peak intensity, chromatographic separation, and the degree of HSA sequence coverage (Table 1).
Table 2. Peak Area Ratio Repeatability for the Buffer and Urine Systemsa buffer system
sample Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa
Precision Equivalence of Multiplexed Urine Albumin Assay
Measurement reproducibility, sensitivity, and selectivity are critical factors that affect the accuracy and validity of MS-based quantitative assays. Therefore, to assess the repeatability and measurement equivalence of the 11 peptides chosen for the optimized multiplexed assay, we spiked a known ratio of unlabeled-to-labeled rHSA in a buffer (50 mmol/L ammonium bicarbonate in water) system and control human urine (NIST SRM 3667) system and calculated the average peak area ratio (unlabeled peak area/15N-labeled peak area) of each MRM transition for three technical replicates (Table 2). To correct the peak area ratios for the endogenous albumin present in SRM 3667, the endogenous unlabeled-to-labeled rHSA peak area ratio of each MRM transition was subtracted from each corresponding MRM transition peak area ratio of the spiked urine samples. The MRM peak profile for both the buffer and urine systems is consistent across the technical replicates and between the two systems (Figure 2). Similarity in the peak area ratios for the buffer and urine systems suggests that there is no major effect of the urine matrix on the MRM peak area ratio for each transition (Table 2). However, the reduced peak area ratios observed in peptide 15 (RPCFSALEVDETYVPK) and peptide 16 (LVAASQAALGL) for both systems could suggest a correlation between digestion efficiency and the peak area ratio. Peptide 15 contains one known missed cleavage at the Nterminus due to the presence of the proline residue, and there are two lysine residues that precede the sequence of peptide 16, which implies that a missed cleavage could potentially alter the m/z of peptide 16 by one lysine residue. The ion currents of peptides containing missed cleavages is comparatively lower than those of fully tryptic peptides; therefore, the reduced peak area ratio observed in peptides 15 and 16 could be due to the presence of a fully cleaved and missed cleavage site, respectively.24,25 In addition to uniformity in the peak area ratios for both systems, the coefficient of variance (% CV) for each transition, with the exception of peptide 1.2 (buffer system), is ≤1.0%, which supports the high precision and accuracy of the assay. From these observations, we conclude that there are no detectable effects of the urine matrix on the repeatability and precision of the assay and that digestion
1.1 1.2 2.1 2.2 4.1 4.2 5.2 5.4 7.1 7.2 11.1 11.3 12.1 12.3 12.4 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2
peptide
peak area ratio
DLGEENFK DLGEENFK LVNEVTEFAK LVNEVTEFAK LCTVATLR LCTVATLR YLYEIAR YLYEIAR AEFAEVSK AEFAEVSK TYETTLEK TYETTLEK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL
3.52 3.46 3.16 3.34 3.51 3.63 3.40 3.41 3.27 3.21 3.53 3.67 3.60 3.79 4.07 3.26 3.66 3.56 3.19 2.49 2.40 2.48 2.59
b
urine (SRM 3667) systemc
% CV
peak area ratio
% CV
0.4 2.3 0.6 0.9 0.3 0.1 0.2 0.5 0.5 0.2 0.4 0.4 0.6 0.6 0.3 0.1 0.1 0.5 0.2 0.9 0.3 0.2 0.4
3.29 3.16 3.20 3.40 3.59 3.74 3.45 3.50 3.18 3.11 3.29 3.44 3.67 3.89 4.24 3.23 3.67 3.61 3.22 2.74 2.60 2.54 2.68
0.4 0.5 0.7 0.4 0.5 0.6 0.2 0.7 0.3 0.5 0.1 0.4 0.1 0.4 0.2 0.8 0.3 1.0 0.6 0.5 0.8 0.1 0.3
a n = 3. bExpected peak area ratio for the buffer (50 mM AMBIC) system, 3.49. cExpected peak area ratio for the urine system, 3.45.
efficiency plays a major role in peptide quantitation. This also supports the need for the multiplexed urine albumin assay because by assessing multiple peptides we are able to clearly detect intra- or interpeptide measurement differences, which can affect the overall quantification of intact albumin in urine. To further assess the equivalence and repeatability of the multiplexed MRM assay, we investigated the standard curve samples in control human urine (NIST SRM 3667). To generate the five urine albumin calibration solutions, unlabeled rHSA (≈5.3, 32.2, 102.7, 193.9, and 308.3 mg/L) and 15Nlabeled rHSA (≈23.1 mg/L) were spiked into the urine matrix (NIST SRM 3667). The actual concentrations (gravimetric measurements) of unlabeled and 15N-labeled material in each calibration set are outlined in Table 3, and the unlabeled rHSA concentrations were selected on the basis of the three clinical urine albumin ranges: normoalbuminuria (0−30 mg/L), microalbuminuria (30−300 mg/L), and macroalbuminuria (>300 mg/L).5 Three experimental replicates of each set of calibrators were prepared and analyzed in triplicate (technical replicates) to assess variability in the proteolytic digestion of the calibrator sets and to evaluate technical precision, repeatability, and peptide equivalence. As shown in Figures 3 and 4 and Table 4, the peak area ratios for each set of calibrators is consistent within and between each peptide, and, although there are slight differences in the actual concentration of unlabeled-to-labeled material between each set, the peak area ratios are repeatable across the 3 day study with high precision (% CV ≤ 1.5, except for four transitions). This data validates the equivalence and repeatability of the multiplexed assay. 3933
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Figure 2. Reproducibility of the multiplexed urine albumin assay peptide profile in buffer (50 mmol/L ammonium bicarbonate) and urine (NIST SRM 3667) systems.
sample, which could suggest heterogeneity in the urine albumin species of the pooled patient material. The average calculated urine albumin content for 9 peptides (excluding peptides 15 and 16) was 157.2 mg/L (% CV 2.2) for day 1, 151.6 mg/L (% CV 1.7) for day 2, and 163.8 mg/L (% CV 2.2) for day 3 (Table 5). To further validate the urine albumin assay, individual patient urine samples were also analyzed to assess the influence of biological variability on the precision of the multiplexed assay. The albumin content of the individual samples was measured via an immunoturbidimetry assay at various levels, within the normoalbuminuria (0−30 mg/L) and microalbuminuria (30− 300 mg/L) clinical ranges (Table 6). Control microalbumin calibrators (controls 1 and 2) were also included in the analysis to monitor the precision of the assay (Table 6). Similar to that in the pooled patient sample, there was an increase in the albumin content of the individual and control urine samples for peptide 15, which could be attributed to the following: the presence of C-terminal albumin fragments or the presence of a modification on the endogenous peptide, which would make the peak area for the patient sample higher than that of the internal standard. Therefore, the albumin measurements from peptide 15 were excluded from the final urine albumin content, which represents an average of the 10 HSA peptides (Table 6). As shown in Table 6, the majority of the individual and the control urine samples are within the expected urine albumin range with the exception of six samples (samples 2, 5, 8, 11, 13, and 14). The inconsistencies between the expected and observed urine albumin measurements are mainly due to differences in the albumin content of the MRM peptides, which suggests the presence of molecular heterogeneity of the albumin species in the individual urine samples as a result of protein truncation or post-translational modifications. Moreover, the % CV values for the averaged urine albumin measurements are below 10%, with the exception of samples 1, 7, and 12, which is due to low % CV values observed in the individual MRM transitions measurements for each sample. By utilizing the multiplexed assay to evaluate the urine albumin content of the pooled and individual urine patient samples, we
Table 3. Standard Calibrators for Urine Albumin Quantitationa calibration set 1
2
3
a
quant levels Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant Quant
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
unlabeled rHSA (mg/L)
15 N-labeled rHSA (mg/L)
concentration ratio
5.3 32.2 102.7 193.9 308.3 6.7 32.5 100.8 193.7 308.4 6.8 33.4 97.7 193.9 307.6
21.0 22.2 23.6 22.1 21.3 22.8 22.4 23.1 23.1 21.9 22.4 22.9 23.3 21.4 21.9
0.3 1.4 4.3 8.8 14.5 0.3 1.5 4.4 8.4 14.1 0.3 1.5 4.2 9.1 14.1
Three day study.
Validation of Multiplexed Urine Albumin Assay
To assess the precision and accuracy of the multiplexed urine albumin assay, we investigated the determination of urine albumin content in a pooled patient urine sample and individual patient urine samples. The pooled material was obtained from the Mayo Clinic, and, via an immunoturbidimetry assay, the albumin content was measured at approximately 160 mg/L, within the microalbuminuria clinical range. Using the urine calibrators, the average calculated urine albumin content for all 11 peptides was 171.8 mg/L (% CV 20.4) for day 1, 165.5 mg/L (% CV 20.0) for day 2, and 178.4 mg/L (% CV 19.5) for day 3 (Table 5). The precision for the calculated albumin measurements for each day is elevated for the pooled sample compared to the % CV values observed in the buffer and urine studies. This increase is due to the increase in the calculated albumin content for peptides 15 and 16 over the 3 day study. This could be due to the presence of C-terminal albumin fragments present only in the pooled patient urine 3934
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Figure 3. Repeatability of the multiplex urine albumin assay peptide profile of quantitative calibrators (Quant Levels 1−5). (Quantitation levels are detailed in Table 3.)
Figure 4. Linear regression analysis of concentration ratio vs MRM peak area ratio for each MRM transition at each quantitation level. The concentration ratio represents the unlabeled-to-labeled albumin concentration ratio and the MRM peak area ratio represents the unlabeled-to-labeled peak area ratio. 3935
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Table 4. Peak Area Ratio Repeatability for Urine Albumin Quantitationa quant level 1 day 1
2
3
sample Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa
1.1 1.2 2.1 2.2 4.1 4.2 5.2 5.4 7.1 7.2 11.1 11.3 12.1 12.3 12.4 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2 1.1 1.2 2.1 2.2 4.1 4.2 5.2 5.4 7.1 7.2 11.1 11.3 12.1 12.3 12.4 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2 1.1 1.2 2.1 2.2 4.1 4.2 5.2 5.4 7.1 7.2 11.1 11.3 12.1
quant level 2
quant level 3
quant level 4
quant level 5
peptide
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
DLGEENFK DLGEENFK LVNEVTEFAK LVNEVTEFAK LCTVATLR LCTVATLR YLYEIAR YLYEIAR AEFAEVSK AEFAEVSK TYETTLEK TYETTLEK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL DLGEENFK DLGEENFK LVNEVTEFAK LVNEVTEFAK LCTVATLR LCTVATLR YLYEIAR YLYEIAR AEFAEVSK AEFAEVSK TYETTLEK TYETTLEK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL DLGEENFK DLGEENFK LVNEVTEFAK LVNEVTEFAK LCTVATLR LCTVATLR YLYEIAR YLYEIAR AEFAEVSK AEFAEVSK TYETTLEK TYETTLEK VFDEFKPLVEEPQNLIK
0.39 0.38 0.36 0.38 0.40 0.41 0.39 0.39 0.38 0.37 0.40 0.42 0.41 0.43 0.46 0.37 0.41 0.40 0.36 0.36 0.34 0.32 0.33 0.31 0.30 0.27 0.29 0.30 0.31 0.29 0.29 0.28 0.27 0.30 0.31 0.30 0.32 0.34 0.27 0.31 0.30 0.27 0.21 0.20 0.20 0.21 0.32 0.29 0.28 0.30 0.31 0.32 0.30 0.30 0.29 0.29 0.31 0.31 0.31
0.5 1.5 0.4 1.5 0.4 0.2 0.3 1.5 0.5 0.4 0.2 0.8 0.7 0.3 1.0 0.6 0.1 0.4 0.3 1.6 1.2 0.2 0.2 0.7 1.8 0.2 1.2 0.1 0.6 0.5 0.2 0.4 0.4 0.2 0.6 0.8 0.5 0.5 0.4 0.4 0.6 0.2 1.3 0.5 0.5 0.5 1.4 0.4 0.8 0.9 0.2 0.3 0.2 0.4 1.0 0.2 0.4 0.7 0.4
1.28 1.25 1.20 1.26 1.33 1.37 1.29 1.30 1.23 1.21 1.30 1.35 1.36 1.43 1.53 1.22 1.36 1.33 1.20 0.95 0.90 0.93 0.97 1.44 1.39 1.32 1.41 1.46 1.51 1.43 1.43 1.38 1.34 1.45 1.51 1.48 1.58 1.68 1.34 1.50 1.47 1.32 1.05 1.01 1.03 1.08 1.33 1.29 1.22 1.28 1.34 1.37 1.30 1.31 1.26 1.24 1.35 1.39 1.38
0.2 2.0 0.2 0.8 0.3 0.8 0.5 0.6 0.2 0.1 0.3 0.7 1.4 0.2 0.9 0.3 0.8 0.4 0.3 0.8 0.7 0.0 0.2 1.2 0.2 0.7 0.2 0.4 0.3 0.4 0.7 0.2 0.6 0.3 0.4 0.9 0.4 0.7 0.6 0.3 0.6 0.4 0.4 0.5 0.1 0.4 0.4 0.9 0.6 0.5 0.3 0.5 0.4 0.7 0.6 0.2 0.5 0.8 1.2
4.30 4.15 4.21 4.49 4.66 4.80 4.51 4.54 4.20 4.13 4.35 4.47 4.73 5.00 5.43 4.21 4.74 4.67 4.21 3.33 3.21 3.22 3.36 4.23 4.07 4.15 4.42 4.59 4.72 4.44 4.44 4.18 4.09 4.31 4.42 4.63 4.92 5.36 4.15 4.70 4.61 4.17 3.29 3.16 3.18 3.31 3.95 3.80 3.78 3.99 4.19 4.34 4.05 4.06 3.83 3.76 4.06 4.18 4.28
0.3 0.6 0.5 0.2 0.4 0.4 0.6 1.7 0.1 0.3 0.9 1.1 1.0 0.0 0.5 0.1 0.3 0.8 1.2 2.0 0.6 0.2 0.3 1.1 1.0 0.4 1.0 0.6 0.7 0.3 0.7 0.2 0.3 0.5 0.6 0.7 0.6 1.4 0.3 0.5 0.7 0.7 0.4 0.6 0.2 0.3 1.6 0.8 0.8 0.4 0.2 0.6 0.3 0.5 0.7 0.3 0.4 1.4 0.2
8.23 7.84 7.59 8.08 8.42 8.62 8.10 8.12 7.79 7.67 8.37 8.57 8.47 8.99 9.76 7.72 8.60 8.35 7.59 6.02 5.71 5.82 6.05 7.46 7.15 6.91 7.34 7.77 7.96 7.48 7.46 7.13 7.03 7.64 7.85 7.74 8.24 9.02 7.05 7.93 7.69 6.96 5.53 5.24 5.36 5.59 7.60 7.34 7.12 7.44 7.89 8.13 7.60 7.70 7.27 7.12 7.77 7.98 7.97
1.5 1.6 0.5 1.5 0.2 0.6 0.3 1.0 0.2 0.7 0.5 0.3 1.9 0.9 0.8 0.3 0.3 0.7 0.5 1.9 0.1 0.5 0.2 1.2 0.9 0.7 0.3 0.3 0.4 0.2 1.5 0.6 0.5 0.6 1.1 1.2 0.9 0.2 0.5 0.3 0.4 1.0 0.7 1.4 0.2 0.8 1.0 0.6 0.9 0.5 0.3 0.6 0.1 0.2 0.2 0.6 0.0 0.7 0.9
12.98 12.33 12.65 13.32 14.00 14.35 13.53 13.45 12.68 12.49 13.38 13.83 13.92 14.90 16.21 12.68 14.35 13.71 12.63 9.82 9.41 9.64 9.96 13.32 12.83 12.63 13.29 13.92 14.16 13.60 13.46 13.00 12.78 13.94 14.23 14.02 14.99 16.30 12.85 14.49 13.90 12.73 9.87 9.44 9.67 10.05 12.68 12.23 12.36 13.03 13.62 13.94 13.16 13.09 12.42 12.12 13.01 13.29 13.65
0.8 1.2 1.1 1.0 0.3 0.3 0.4 0.8 0.4 0.5 1.6 1.0 1.5 0.7 0.3 1.4 0.2 0.4 0.8 2.1 0.4 0.4 0.5 1.9 0.2 0.7 0.6 0.2 0.2 0.5 0.3 1.2 0.2 0.8 1.7 0.6 0.8 1.2 0.9 0.6 0.1 0.8 0.8 0.8 0.1 1.4 0.2 1.4 1.0 0.7 0.5 1.4 0.3 0.8 1.3 0.4 0.5 0.9 0.5
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Table 4. continued quant level 1 day
a
12.3 12.4 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2
quant level 3
quant level 4
quant level 5
peptide
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
peak area ratio
% CV
VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL
0.33 0.36 0.28 0.32 0.32 0.28 0.22 0.21 0.21 0.22
0.3 0.5 0.3 0.2 0.3 0.1 0.7 0.7 0.3 0.6
1.45 1.56 1.23 1.39 1.35 1.21 0.95 0.91 0.94 0.98
0.8 0.8 0.7 0.2 0.7 0.2 0.7 0.8 0.1 0.1
4.49 4.87 3.80 4.26 4.17 3.78 2.95 2.85 2.89 3.03
0.5 1.1 0.2 0.3 0.3 0.4 2.1 1.1 0.5 0.4
8.44 9.12 7.18 8.09 7.83 7.09 5.58 5.39 5.47 5.68
0.2 0.9 1.3 0.7 0.5 0.6 0.5 1.2 0.1 0.7
14.65 15.85 12.41 13.97 13.42 12.36 9.61 9.29 9.44 9.89
0.5 0.9 0.4 0.6 0.8 0.6 1.6 0.6 0.3 0.9
sample Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa
quant level 2
Three day study; n = 3..
Table 5. Determination of Urine Albumin Content for the Pooled Patient Samplea day 1 sample Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa Hsa
a
1.1 1.2 2.1 2.2 31.1 31.2 5.2 5.4 7.1 7.2 11.1 11.3 12.1 12.3 12.4 13.1 13.3 14.1 14.2 15.1 15.2 16.1 16.2
day 2
day 3
peptide
albumin content (mg/L)
% CV
albumin content (mg/L)
% CV
albumin content (mg/L)
% CV
DLGEENFK DLGEENFK LVNEVTEFAK LVNEVTEFAK LCTVATLR LCTVATLR YLYEIAR YLYEIAR AEFAEVSK AEFAEVSK TYETTLEK TYETTLEK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK VFDEFKPLVEEPQNLIK FQNALLVR FQNALLVR QTALVELVK QTALVELVK RPCFSALEVDETYVPK RPCFSALEVDETYVPK LVAASQAALGL LVAASQAALGL average albumin content (mg/L)b average albumin content (mg/L)c
158.78 160.25 154.67 151.79 155.38 155.20 156.04 156.33 157.22 155.79 164.69 164.39 155.20 154.11 152.36 157.82 158.13 159.31 158.72 268.46 272.04 213.14 212.60 171.84 157.17
0.9 1.3 0.1 0.3 0.3 0.3 0.1 1.1 0.6 0.1 0.3 1.4 0.6 0.1 1.1 0.6 0.9 0.4 0.6 0.7 0.3 0.1 0.5 20.4 2.2
152.19 152.65 149.20 150.47 150.24 150.95 151.26 151.75 150.38 150.31 156.58 159.08 149.88 149.16 147.16 152.48 151.66 153.15 152.12 255.63 261.32 204.96 204.40 165.52 151.61
0.7 2.5 0.7 0.7 0.5 0.6 0.3 0.6 0.4 0.4 0.5 0.8 2.6 0.4 0.8 0.6 0.7 0.7 0.6 1.4 1.0 0.2 0.5 20.0 1.7
166.30 163.45 161.35 161.22 161.98 160.82 163.38 163.43 164.01 162.86 171.63 172.71 159.78 160.31 160.24 163.35 164.56 167.04 164.15 274.65 276.88 219.89 220.17 178.44 163.82
0.8 1.3 0.3 0.5 0.2 0.4 0.9 1.2 0.4 0.3 0.4 0.9 1.2 0.5 0.9 0.4 0.2 0.6 1.2 0.7 1.0 0.3 0.8 19.5 2.2
n = 3. bAverage albumin content for all 11 peptides. cAverage albumin content for 9 peptides (excludes peptides 15 and 16).
both domains, which affects the precursor ion mass for the initial separation in the mass spectrometer. The usage of immunochemical methods to differentiate the full-length species from albumin fragments could produce biased results due to the accessibility and location of the antibody epitope.
are able to assess the molecular heterogeneity of endogenous urine albumin due to the incorporation of multiple peptides distributed across the amino acid sequence of albumin. In addition to quantification of the pooled and individual patient urine samples, the multiplexed assay was also used to distinguish between the three albumin domains (domains I, II, and III) in a urine system (NIST SRM 3667). As illustrated in Figure 1, a target peptide is present in each domain, with the exception of peptide 12, which is split between domains II and III. Using the multiplexed assay, we are able to differentiate between the full-length albumin species and the three albumin domains based on the absence of specific target peptides in each MRM chromatogram (Figure 5). As stated above, peptide 12 is not present in the MRM chromatogram of the tryptic digest of domain II or III because it is only partially present in
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CONCLUSIONS
The emergence of urinary proteomics as a valuable approach for the characterization of proteins/peptides involved in the pathological processes of renal dysfunction development and progression has led to the usage of proteomics-based platforms in biomedical research. In this study, we introduce a multiplexed candidate reference measurement procedure that utilizes mass spectrometry and an isotopically labeled intact internal standard (15N-labeled rHSA) for the absolute 3937
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species, or N-/C-terminal truncations. Additionally, due to the presence of the labeled internal standard prior to sample preparation, the interferences from sample handling on the quantitative measurements are insignificant. In addition to the intact albumin quantitative advantages, usage of the MS-based MRM technique offers a high degree of analytical specificity and sensitivity to the multiplexed assay. The triple quadrupole mass spectrometer selectively separates the precursor/product ion pair of each target peptide from the bulk digestion products; therefore, the urine sample can be analyzed with minimal sample preparation. As a result of the selective nature of the MRM method, the sensitivity of the assay is enhanced, which enables the detection of urine albumin at the lower normoalbuminuria range (lower limit of quantitation of approximately 5.3 mg/L) with high precision and accuracy. The dynamic range of the multiplexed assay traverses all three albuminuria stages (approximately 5−300 mg/L), and this supports the usage of the assay for early detection of microalbuminuria and the observation of renal dysfunction progression from normoalbuminuria to macroalbuminuria. In addition to the selectivity and sensitivity advantages of the multiplexed assay, the intra- and interpeptide peak area ratios and quantitative measurements are highly comparable and reproducible between experimental replicates and within technical replicates with high precision, as illustrated in Tables 4 and 5. The accuracy and repeatability of the multiplexed urine albumin assay supports its incorporation to the urine albumin reference system as a candidate reference measurement procedure for clinical urine albumin measurements of certified reference and high-order calibration materials and the commutability assessment of such materials.
Table 6. Determination of Urine Albumin Content for Individual Patient Urine Samples individual patient urine sample
expected urine albumin content (mg/L)a
observed urine albumin content (mg/L)b
% CV
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 control 1 control 2
20−40 40−60 40−60 20−40 60−80 30−40 10−20 10−20 40−60 20−30 40−60 10−20 80−100 20−40 20−30 26.3−39.5 47.6
34.52 30.17 49.32 23.02 47.90 30.94 24.02 22.97 44.10 26.16 33.41 18.67 79.06 18.72 23.85 35.29 41.25
12.7 7.4 5.9 9.3 6.2 7.5 10.8 9.7 7.9 8.8 6.9 11.7 4.2 9.0 6.5 7.6 6.1
a
Expected urine albumin content determined from immunoturbidimetric assay. bAverage observed urine albumin content of all MRM peptides except for peptide 15.
quantification of full-length urine albumin. The multiplexed assay takes advantage of the full-length internal standard by assessing multiple peptides that span the amino acid sequence of mature albumin (loss of N-terminal signal sequence) (Figure 1). This supports the absolute quantification of intact urine albumin, and any alterations observed in the peak area ratio (unlabeled-to-labeled) of the targeted peptides could suggest the presence of albumin fragments, splice variants, modified
Figure 5. Chromatographic profile of human serum albumin domain differentiation using the multiplexed LC−MS/MS urine albumin assay with 11 peptides in full-length unlabeled recombinant human serum albumin (rHSA) and unlabeled rHSA domains I, II, and III. 3938
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cleavage liquid chromatography-tandem mass spectrometry. Clin. Chem. 2009, 55, 1991−1994. (8) Ellam, T. J. Albumin:creatinine ratio−a flawed measure? The merits of estimated albuminuria reporting. Nephron Clin. Pract. 2011, 118, c324−330. (9) Zuo, Y.; Wang, C.; Zhou, J.; Sachdeva, A.; Ruelos, V. C. Simultaneous determination of creatinine and uric acid in human urine by high-performance liquid chromatography. Anal. Sci. 2008, 24, 1589−1592. (10) Martin, H. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease. Clin. Biochem. Rev. 2011, 32, 97−102. (11) Miller, W. G.; Bruns, D. E.; Hortin, G. L.; Sandberg, S.; Aakre, K. M.; McQueen, M. J.; Itoh, Y.; Lieske, J. C.; Seccombe, D. W.; Jones, G.; Bunk, D. M.; Curhan, G. C.; Narva, A. S.; National Kidney Disease Education Program-IFCC Working Group on Standardization of Albumin in Urine. Current issues in measurement and reporting of urine albumin excretion. Clin. Chem. 2009, 55, 24−38. (12) Sviridov, D.; Drake, S. K.; Hortin, G. L. Reactivity of urine albumin (mircoalbumin) assays with fragmented or modified albumin. Clin. Chem. 2008, 54, 61−68. (13) Barnaby, O. S.; Cerny, R. L.; Clarke, W.; Hage, D. S. Quantitative analysis of glycation patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS. Clin. Chim. Acta 2011, 412, 1606−1615. (14) Brennan, S. O.; George, P. M. Three truncated forms of serum albumin associated with pancreatic pseudocyst. Biochim. Biophys. Acta 2000, 1481, 337−343. (15) Cha, T.; Tahara, Y.; Yamato, E.; Yoneda, H.; Ikegami, H.; Noma, Y.; Shima, K.; Ogihara, T. Renal handling of glycated albumin in non-insulin-dependent diabetes mellitus with nephropathy. Diabetes Res. Clin. Pract. 1991, 12, 149−156. (16) Bar-Or, D.; Rael, L. T.; Bar-Or, R.; Slone, D. S.; Craun, M. L. The formation and rapid clearance of a truncated albumin species in a critically ill patient. Clin. Chim. Acta 2006, 365, 346−349. (17) Wiggins, R. S.; Kshrisagar, B.; Kelsch, R. C.; Wilson, B. S. Fragmentation and polymeric complexes of albumin in human urine. Clin. Chim. Acta 1985, 149, 155−163. (18) Candiano, G.; Musante, L.; Brushi, M.; Petretto, A.; Santucci, L.; Del Boccio, P.; Pavone, B.; Perfumo, F.; Urbani, A.; Scolari, F.; Ghiggeri, G. M. Repetitive fragmentation products of albumin and alpha1-antitrypsin in glomerular diseases associated with nephrotic syndrome. J. Am. Soc. Nephrol. 2006, 17, 3139−3148. (19) Pan, S.; Aebersold, R.; Chen, R.; Rush, J.; Goodlett, D. R.; McIntosh, M. W.; Zhang, J.; Brentnall, T. A. Mass spectrometry based targeted protein quantification: methods and applications. J. Proteome Res. 2009, 8, 787−797. (20) Brun, V.; Masselon, C.; Garin, J.; Dupuis, A. Isotope dilution strategies for absolute quantitative proteomics. J. Proteomics 2009, 72, 740−749. (21) Copeland, B. E.; Grisley, D. W.; Casella, J.; Bailey, H. Comparison of serum calcium measurements with respect to five models of atomic absorption spectrometers using NBS-AACC calcium reference method and isotope-dilution mass spectrometry as the definitive method. Am. J. Clin Pathol. 1976, 66, 619−633. (22) Barr, J. R.; Maggio, V. L.; Patterson, D. G., Jr.; Cooper, G. R.; Henderson, L. O.; Turner, W. E.; Smith, S. J.; Hannon, W. H.; Needham, L. L.; Sampson, E. J. Isotope dilution−mass spectrometric quantification of specific proteins: model application with apolipoprotein A-1. Clin. Chem. 1996, 42, 1676−1682. (23) Bunk, D. Reference materials and reference measurement procedures: an overview from a National Metrology Institute. Clin. Biochem. Rev. 2007, 28, 131−137. (24) Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008, 4, 1−14. (25) Picotti, P.; Aebersold, R.; Domon, B. The implications of proteolytic background for shotgun proteomics. Mol. Cell. Proteomics 2007, 6, 1589−1598.
ASSOCIATED CONTENT
S Supporting Information *
LTQ-XL MS parameters (Table S1), 95 min LC gradient (Table S2), tandem MS peptide identification protein sequence coverage (Table S3), tandem MS peptide identification (Table S4), peak area comparison for peptide states on triple quadrupole MS (Table S5), comparison of peak area for MRM transitions (Table S6), peptide collision energy optimization (Table S7), peptide fragmentor optimization (Table S8), transition dwell time optimization (Table S9), buffer system MRM assay (peak area ratio comparison) (Table S10), buffer system MRM assay results (Table S11), urine system MRM assay (peak area ratio comparison) (Table S12), urine system MRM assay results (Table S13), tandem MS base peak chromatogram of unlabeled and 15N-labeled rHSA (Figure S1), tandem MS spectra of selected peptides (Figure S2), peak intensity bar graph of potential MRM transitions (Figure S3), and bar graph of retention time for each potential MRM transition (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: (301) 977-0685. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS A special thanks to Dr. Illarion Turko for the production of the 15 N-labeled recombinant HSA material used in this project. We also thank Dr. John Lieske at the Mayo Clinic for his kind donation of patient urine samples.
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ABBREVIATIONS mass spectrometry, MS; liquid chromatography−tandem mass spectrometry, LC−MS/MS; isotope dilution−mass spectrometry, ID−MS; multiple reaction monitoring, MRM; albumin− creatinine ratio, ACR; collision energy, CE; coefficient of variance, CV; National Institute of Standards and Technology, NIST; National Kidney Disease Education Program, NKDEP; Working Group for the Standardization of Albumin Assays in Urine, WG-SAU; of the International Federation of Clinical Chemistry, IFCC
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REFERENCES
(1) Kestenbaum, B.; de Boer, I. H. Urine albumin-to-creatinine ratio: what’s in a number? J. Am. Soc. Nephrol. 2010, 21, 1243−1244. (2) Decramer, S.; Gonzalez, de; Peredo, A.; Breuil, B.; Mischak, H.; Monsarrat, B.; Bascands, J. L.; Schanstra, J. P. Urine in clinical proteomics. Mol. Cell Proteomics. 2008, 7, 1850−1862. (3) Danziger, J. Importance of low-grade albuminuria. Mayo Clin. Proc. 2008, 83, 806−812. (4) Peters, T. Jr. All About Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (5) de Jong, P. E.; Gansevoort, R. T.; Bakker, S. J. Macroalbuminuria and microalbuminuria: do both predict renal and cardiovascular events with similar strength? J. Nephrol. 2007, 20, 375−80. (6) Choi, S.; Choi, E. Y.; Kim, H. S.; Oh, S. W. On-site quantification of human urine albumin by a fluorescence immunoassay. Clin. Chem. 2004, 50, 1052−1055. (7) Seegmiller, J. C.; Sviridov, D.; Larson, T. S.; Borland, T. M.; Hortin, G. L.; Lieske, J. C. Comparison of urine albumin quantification by immunoturbidimetry, competitive immunoassay, and protein3939
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