Surface-enhanced laser desorption time-of-flight mass spectroscopy (SELDI-TOF-MS) is a bioanalytical technique used for the rapid examination of intact protein or protein mixtures to exploit the biochemical or biophysical characteristics of intact molecules to separate a complex protein mixture or isolate specific protein classes. Surface-enhanced laser desorption time-of-flight mass spectroscopy allows for rapid examination of protein components of body fluids or cell lysates without extensive extraction or preparative measures. The levels and composition of proteins found in blood and urine may change after exposure to toxic agents.1,2 Such potential changes make the proteins found in these easily obtained fluids a desirable source of new or altered proteins that indicate toxic exposure. This article describes the use of SELDI-TOF to examine urinary proteins or hemoglobin present in erythrocyte lysates.
Acrylamide is a widely used industrial chemical intermediate with many applications,3 such as a polymerizing agent in grouts and in the preparation of laboratory gels for protein and nucleic acid electrophoresis. Low levels of acrylamide present in baked, fried, and roasted foods and in tobacco smoke are common sources of human exposure.4 Acrylamide is a potent neurotoxin5 and is a probable human carcinogen that makes exposure a concern for human health.6
Figure 1 - Acrylamide metabolism and hemoglobin adduct formation. Acrylamide is metabolized to glycidamide by a detoxifying enzyme Cytochrome P450 CYP2E1 found in liver, lung, and brain tissue.
Acrylamide and its metabolite glycidamide, also considered a toxicant, are reactive compounds and readily form adducts with biological macromolecules, including proteins.7,8 Both acrylamide and glycidamide react with hemoglobin (HGB), specifically at the N-(2 carbamoylethyl) valine residue of the β-peptide subunit (Figure 1). These valine adducts are considered a biomarker of long-term acrylamide exposure.3 Possible reactions of acrylamide and glycidamide with other amino acids suggest that urinary proteins may be affected in acrylamide exposure.9 One of the goals of this work was to identify proteins in human urine modified by acrylamide exposure using SELDI-TOF-MS and four types of ProteinChip® array (Bio-Rad Laboratories, Hercules, CA) to determine which array would be most useful for the planned analysis of urine from occupationally exposed workers. A second goal was to evaluate SELDI-TOF-MS as a low-cost, rapid screening method to demonstrate acrylamide or glycidamide adducted hemoglobin.
Methods
Urinary protein profiling
Control urine specimens were collected from healthy male volunteers working at the National Institute for Occupational Safety and Health (NIOSH) with no smoking history or occupational exposure (n = 16). End-of-work-shift samples were collected at an acrylamide production plant from nonsmoking, occupationally exposed workers (n = 34); smoking, occupationally exposed workers (n = 24); and nonsmoking, nonoccupationally exposed office workers (n = 12). Only nonsmoking office workers were selected as controls. The acrylamide workers study was approved by the NIOSH Human Subjects Review Board.
Samples collected at the production site were frozen and shipped to NIOSH and subjected to one freeze–thaw cycle prior to extraction and analysis. Urine samples were prepared and extracted using previously described methods.10 Briefly, urine samples were prepared by centrifugation at 16,000 × g for 5 min at 4 °C to sediment cellular material prior to use. Urinary proteins were extracted from a 160-μL aliquot of each sample by mixing with 60 μL denaturing buffer (9 M urea/2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS)/50 mM Tris, pH 9.0), vortexed for 30 min at 4 °C, and then centrifuged for 1 min at 5000 × g before analysis of the supernatant in triplicate. For incubations of urine with either acrylamide or glycidamide, freshly collected urine was filtered through a 0.2-μm pore size cellulose acetate filter to remove urinary sediment and bacteria. Aliquots of urine were incubated for 24, 48, 72, and 96 hr in the presence of 10 mM acrylamide or glycidamide in sterile glass screw-cap tubes placed in an oscillating water bath at 37 °C. Samples were extracted as described above and then applied to IMAC30 ProteinChip arrays for SELDI-TOF analysis.
SELDI-TOF analysis of urinary proteins
Table 1 - ProteinChip® characteristics and preparation protocols
Figure 2 - Process flow in SELDI-TOF-MS. 1) A complex protein mixture is applied to a ProteinChip. 2) Nonspecifically bound proteins are washed away. 3) Energy-absorbing matrix is applied to all samples, and a laser beam desorbs and ionizes the protein crystallized in the matrix. 4) Ions are captured and the mass of each protein is calculated by TOF-MS.
Urinary proteins were processed on NP20, CM10, H50, and IMAC30 chip arrays (Bio-Rad) having normal-phase, cationic- exchange, hydrophobic, and ionic surface chemistries, as described in Table 1. Various factors were optimized during the development of binding protocols for each chip, including chip surface pretreatment, sample binding time, and washing methods (see Table 1). Figure 2 illustrates sample preparation and process flow. ProteinChip arrays were analyzed by SELDI-TOF-MS with a Ciphergen PBS II Reader (Bio-Rad) using the following conditions in positive mode: laser intensity, 150; detector sensitivity, 9; detector voltage, 1700 V; spectra average, 240/sample; optimization range, 500–12,500 Da for urinary protein detection; calibration, external mass accuracy using a ProteinChip QC Peptide Array, MW range 7–147 kDa. Mass spectra baseline correction, normalization, peak detection, and alignment were performed using either Ciphergen ProteinChip software (version 3.0.2) or R language (version 2.0.1)11 functions implemented in the PROcess library of the BioConductor Project (version 1.5).12 The number of peaks and mean peak intensity varied significantly with chip type, where the number and intensity of peaks was greatest using IMAC30 > CM10 > NP20 > H50.
Erythrocyte incubation with acrylamide or glycidamide
Whole human blood specimens were collected by venipuncture into ethylenediaminetetraacetic acid (EDTA) containing Vacutainers (Becton Dickinson, Franklin Lakes, NJ). Blood was separated into erythrocytes and plasma by centrifugation. Erythrocytes were isolated and washed three times with physiological saline solution. To examine native hemoglobin, washed erythrocytes were lysed by adding one volume of distilled water, and the lysate was immediately processed for SELDI-TOF analysis. To produce adducted hemoglobin, washed erythrocytes were suspended in an equal volume of RPMI 1640 medium base with 1% penicillin-streptomycin solution with or without 10 mM acrylamide or glycidamide. Erythrocyte cultures were incubated at 37 °C in a 5% CO2 incubator. Cultures were sampled at 24, 48, 72, and 96 hr incubation.
Cultured erythrocytes were washed with saline and lysed as described above. After centrifugation to remove cell membranes and cellular debris, hemoglobin samples were used immediately in SELDI-TOF analysis. Hemoglobin samples were processed on H50 ProteinChip arrays with a Ciphergen PBS II ProteinChip reader using instrument conditions similar to those described above in urinary protein analyses. SELDI-TOF analysis was performed as described above for urinary proteins using a 14–17 kDa optimization range for globin adduct detection.
Results and discussion
Initial SELDI-TOF analyses of human urine showed that urine specimens collected from healthy male volunteers working at NIOSH with no smoking history or occupational exposure produce complex and highly variable SELDI spectra. The variations observed in these spectra, possibly due to individual dietary habits including coffee and fried food consumption, complicated the interpretation of spectra in a search for an indicator of acrylamide exposure for use in evaluating occupational exposure. Subsequent SELDI-TOF analysis of urine collected from nonoccupationally exposed, nonsmoking office workers; occupationally exposed acrylamide production plant workers; and occupationally exposed smokers also yielded equally complex and variable spectra.
Exposure to acrylamide dust in production plants may occur through intact skin or by breathing the dust produced by handling and packaging of product pellets or dust formed by sublimation of the solid into dust vapor. Because acrylamide may be metabolized into glycidamide in lung tissue, it was necessary to investigate the possible contribution of smoking to the alteration of urinary proteins. To assist in identifying urinary proteins altered by acrylamide exposure via tobacco smoke, urine collected from a healthy nonsmoking control volunteer with no occupational exposure was examined and compared to replicate urine incubated with acrylamide or glycidamide. In the same manner, replicate urine samples from a nonoccupationally exposed smoker were incubated with acrylamide or glycidamide.