Bioconjugate Chem. 1996, 7, 475−481
475
Characterization of Protein-Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin-Hapten Conjugates† Maciej Adamczyk,* John C. Gebler, and Phillip G. Mattingly Abbott Laboratories, Diagnostics Division, Division Organic Chemistry (9-NM), Building AP 20, 100 Abbott Park Road, Abbott Park, Illinois 60064. Received March 12, 1996X
Fifteen hapten-bovine serum albumin (BSA) conjugates were prepared from five commercially available activated haptens. Each hapten was coupled to BSA at three different ratios. The conjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two mass spectrometry (MS) methods: matrix-assisted laser desorption ionization (MALDI) and liquid chromatography-electrospray ionization (LC-ESI). SDS-PAGE was useful in detecting protein crosslinking, but not in assessing hapten density. MALDI-MS and LC-ESI-MS gave comparable qualitative results, but LC-ESI-MS provided a clearer representation of the distribution of hapten-protein species present in the conjugates. Conjugate species substituted with up to 25 haptens per BSA were recorded by LC-ESI-MS.
INTRODUCTION1
The chemical derivatization of proteins to form bioconjugates is an extremely important process in the field of biotechnology. Still, the characterization of such bioconjugates is a challenging problem. Several methods have been used to estimate the degree of protein derivatization. Direct UV absorbance measurements have been used when the max of the introduced chromophore is distinct from that of the protein (1). UV difference spectroscopy provided an alternative method when there was spectral overlap (2). When the derivatizing agent is doped with a radiolabeled analog, the extent of modification can be followed directly by measuring the associated radioactivity (3). Assay for the unreacted amino groups on the protein either indirectly, by TNBS titration (4-6), or directly, by amino acid analysis (710), is an additional method of bioconjugate characterization. Another means of bioconjugate characterization relies on the measurement of the molecular weight of the modified protein. Until recently, only low-resolution methods such as SDS-PAGE (11) or isoelectric focusing (1, 12) were available. Now, mass spectrometry techniques (FAB, MALDI, ESI) have emerged as powerful tools for directly observing the mass of modified proteins (13, 14) and thereby offer a means of quantifying the extent and type of modification (15, 16). Recently, we compared MALDI-MS with other more traditional methods for the characterization of a series of conjugates (17). At that time, MALDI-MS appeared to be the method of choice for the direct observation of the average molecular weight of the derivatized protein. In some cases, sub†
In memory of Dr. Roger L. Boeckx, 1946-1995. * Telephone: (847) 937-0225. Fax: (847)938-8927. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, July 1, 1996. 1 Abbreviations: BSA, bovine serum albumin; CZE, capillary zone electrophoresis; DCC, N,N-dicyclohexylcarbodiimide; DMF, dimethylformamide; EDAC, N-ethyl-N-[3-(dimethylamino)propyl]carbodiimide hydrochloride; ESI, electrospray ionization; FAB, fast atom bombardment; HAP, hapten; HPLC, highperformance liquid chromatography; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionization; NHS, Nhydroxysuccinimide; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TNBS, trinitrobenzenesulfonic acid.
S1043-1802(96)00035-3 CCC: $12.00
populations of the conjugate which differed in molecular weight were observed, but the limited resolution of MALDI-MS precluded more detailed analysis. Electrospray ionization mass spectrometry (ESI-MS) is a soft ionization technique which typically gives only the parent ion with little or no fragmentation under normal conditions (14, 18). Unlike MALDI-MS, samples are introduced as solutions in water, organic solvents, or a combination of the two. This is particularly convenient for the analysis of proteins. In addition to direct sample introduction, the use of solutions further allows HPLC or CZE to be interfaced with the mass spectrometer. Regardless of the interface, samples are sprayed into the first chamber of the ESI-MS instrument as a fine mist at atmospheric pressure through a metal needle. Variable ionization potential is applied to the needle, and the charge is transferred to the exiting fine droplets. As the droplets evaporate, the charge is transferred from the solvent to the analyte. The charged analyte enters the high vacuum compartment through an orifice located at the end of the first chamber and is subsequently mass analyzed. Modern instruments are capable of mass determination of molecules ranging from small, simple organics (30 Da) to proteins (up to 200 kDa) with an accuracy of greater than 0.01% (19). Thus, ESI-MS is becoming a common tool for molecular weight determination of proteins, DNA/RNA (20-22), peptides, and other macromolecules. However, there have been limited reports on characterization of conjugates using ESI-MS. A recent report detailed the characterization of haptenenzyme conjugates and concluded that for proteins in the mass range of 55 kDa, only conjugates with a hapten density of eight or less could be analyzed (23). In the present work we have prepared a series of BSA conjugates using five structurally different haptens. Three molar ratios of each activated hapten to BSA were used to achieve different levels of modification in order to study the scope and limitations of ESI-MS as compared with MALDI-MS and SDS-PAGE methods. MATERIALS AND METHODS
The N-hydroxysuccinimide (NHS) esters of 5-carboxyfluorescein (1a), 7-(dimethylamino)-4-coumarinacetic acid (2a), and 3-aminodigoxigenin hemisuccinimide (4a) were obtained from Molecular Probes (Eugene, OR). Dansyl chloride (3a) was purchased from Fluka Chemical © 1996 American Chemical Society
476 Bioconjugate Chem., Vol. 7, No. 4, 1996
(Ronkokoma, NY), and biotin NHS (5a) was obtained from Pierce Chemical (Rockford, IL). BSA (fraction V powder) and Sephadex G-25 Fine were purchased from Sigma Chemical Co. (St. Louis, MO). Anhydrous dimethylformamide (Aldrich Chemical Co., Milwaukee, WI) was vacuum degassed and then used immediately. All other chemicals were of the purest grades commercially available. Solutions were prepared using water from a Millipore water system (Marlborough, MA). Dialysis was performed using the Pierce Slide-A-Lyzer with a 10 kDa molecular weight cutoff and Pierce BupH phosphatebuffered saline (pH 7.2). Analysis by electrospray ionization mass spectrometry was carried out on a PerkinElmer Sciex API 100 Benchtop LC/MS system (Norwalk, CT) employing the Turbo IonSpray interface, interfaced with an binary microbore HPLC system (Ultra Plus, Microtech Scientific, Sunnyvale, CA). Analysis by matrixassisted laser desorption ionization mass spectrometry employed a Bruker Reflex (Bruker Instruments, Billerica, MA) time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm). Gel electrophoresis was performed on a Pharmacia PhastSystem (Piscataway, NJ) utilizing 12.5% polyacrylamide SDS-PAGE precast gels (PhastGel). Gel permeation chromatography was carried out on a Pharmacia P-50 system (Piscataway, NJ) with UV detection at 280 nm. The theoretical average molecular weight of BSA was calculated from the published sequence (24) with the Protein Analysis Worksheet (PAWS) version 5.1 by R. Beavis [mcphar04.med.nyu.edu]. Preparation of Conjugates. Conjugates were prepared at three different ratios of hapten to BSA (5:1, 15: 1, and 30:1). The BSA concentration, the organic solvent content, and the reaction volume were held constant for each preparation. Thus, a stock solution of BSA (300 µM) was prepared by dissolving BSA (40 mg, 0.6 µmol) in phosphate buffer (2 mL, pH 8.0, 50 mM, Fisher Chemical, Fairlawn, NJ). Stock solutions (21 mM) for each of the activated haptens (1a-5a) were prepared by dissolving each hapten (21 µmol) in anhydrous dimethylformamide (1 mL). For each conjugate, the BSA stock solution (0.5 mL, 0.15 µmol) was placed in a vial and treated as follows: (A) For the 5:1 coupling ratio, DMF (179 µL) and the activated hapten (35.8 µL, 0.75 µmol, 500 mol %) were added; (B) For the 15:1 coupling ratio, DMF (107 µL) and the activated hapten (107 µL, 2.27 µmol, 1500 mol %) were added: (C) For the 30:1 coupling ratio, DMF (215 µL) and the activated hapten (215 µL, 4.55 µmol, 3000 mol %) were added. After 4 h, an aliquot (200 µL) from each vial was removed and dialyzed against phosphate buffer with 10% ethanol (500 mL, 2×, 24 h each, then against 10% aqueous ethanol (500 mL, 2×, 4h each), and finally against water (500 mL, 4 h). The remaining vial contents were passed through G-25 column (1 × 20 cm) eluting at 1 mL/min with ammonium acetate buffer (100 mM, 10% methanol). Fractions (3 mL each) were collected, and those for the conjugate peak were pooled. An aliquot (100 µL) of each of the pooled fractions was retained, and the remainder was lyophilized (48 h). Analysis of Conjugates. Each lyophilized conjugate was analyzed by SDS-PAGE, MALDI-MS, and LC-ESIMS. In addition, a LC-ESI-MS spectrum was recorded for each conjugate before purification, after dialysis, and after G-25 gel chromatography. Gel Electrophoresis. Conjugates were dissolved in SDS-PAGE sample buffer (10 mM TRIS/HCl, 1 mM EDTA, 2.5% SDS, and 5.0% β-mercaptoethanol, pH 8.0) to 1 mg/mL and heated to 100 °C for 5 min. Gels were loaded with 2-3 µL of sample and run under denaturing conditions using the manufacturer’s protocol. Upon completion, the gels were stained with Coomassie Blue
Adamczyk et al.
using a standard developing routine from the manufacturer. The resulting gels were evaluated first by scanning (Apple OneScanner, Apple Computers, CA) and second by analyzing the image with the gel-plotting macros of NIH-Image (v 1.58, public domain image processing and analysis program for the Apple Macintosh PowerPC from NIH, Bethesda, MD, downloaded from zippy.nimh.nih.gov). Mass Spectrometry: (A) MALDI-MS. Analysis by matrix-assisted laser desorption ionization mass spectrometry employed a Bruker Reflex (Bruker Instruments, Billerica, MA) time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm). The crystal matrix, sinapinic acid (Aldrich Chemical Co., Milwaukee, WI), was prepared at a concentration of 15 mg/mL in acetonitrile. Protein samples were typically 10-50 pmol/µL in a 2:1 water/acetonitrile solution. Sample and matrix solutions were mixed in equal volumes (typically 1.5 µL each) directly on the stainless steel probe tip (target) and allowed to dry (∼10 min) in a fume hood at room temperature. The crystallized analyte-matrix sample was then rinsed with 0.1% TFA solution by placing approximately 2 µL of the solution on the probe sample at room temperature, allowing it to stand for about 5 s, and then gently drying the crystals with a stream of nitrogen. Spectra were recorded at a threshold laser irradiance for 50-150 shots in the linear mode at 30 kV. The resulting data were analyzed using XMASS, the postacquisition software supplied with the spectrometer (17). (B) LC-ESI-MS. Protein conjugates samples were dissolved in 0.2% formic acid (1 mg/mL) and introduced onto the ESI-MS by HPLC employing a PLRP-S column (2.1 × 50 mm, Polymer Laboratories Ltd., Amherst, MA) using the following linear gradient: 90:10 0.2% formic acid/methanol to 100% methanol over 20 min at a flow of 100 µL/min. Ion source conditions: ion spray (IS) 4800 V, orifice (OR) 30 V, nebulizing gas 1.46 L/min, and TurboProbe jet 350 °C with air flow of 4 L/min. The samples were scanned over two m/z ranges (150-700 and 1200-2400) with a total acquisition time of 7.2 s. Small molecule impurities were detected by reading their molecular weights directly from the low m/z spectra. Molecular weights of the conjugates were determined from the high m/z scans after converting the complex spectra of multicharged protein species to the mass domain using the manufacture’s deconvolution software (BioReconstruct). RESULTS
Conjugation of Haptens. Commercially available, activated haptens (1a-5a) reacted with BSA in pH 8 buffer at three molar ratios (5:1, 15:1, and 30:1) to give the corresponding series of conjugates 1c-5c along with the hydrolyzed haptens 1b-5b (Scheme 1). On the basis of the MS analysis, purification by either gel permeation chromatography or dialysis separated the conjugate from the low molecular weight components. Covalent attachment of each hapten proceeded through the amino groups (59, lysine -amino groups, plus the amino terminal) on the protein with a detectable increase in the molecular weight of the conjugate. The incremental change in molecular weight due to incorporation of the hapten radicals ranged from 227 to 473 (fluorescein 1, ∆MW 359; coumarin 2, ∆MW 230; dansyl 3, ∆MW 234; 3-aminodigoxigenin 4, ∆MW 473; and biotin 5, ∆MW 227). Apparent coupling efficiency to BSA was similar for all the haptens but coumarin 2a, which coupled poorly. All
ESI−MS of BSA−Hapten Conjugates
Bioconjugate Chem., Vol. 7, No. 4, 1996 477
Scheme 1a
Figure 1. (A) Gel electrophoresis of the 5c conjugate series. (B) NIH image digital plot of the 5c conjugate series: (a) phosphorylase b; (b) bovine serum albumin; (c) ovalbumin; (d) carbonic anhydrase; (e) trypsin inhibitor.
a Reaction conditions: pH 8 phosphate buffer/DMF (70:30), 4 h, room temperature.
of the conjugates were completely soluble throughout the conjugation and purification procedures. Analysis of Conjugates. Gel Electrophoresis. The solubility of all conjugate samples allowed for successful electrophoresis. No precipitation of the samples on the gel or during electrophoresis was encountered. Typical results are shown in Figure 1A for the 5c conjugate series. In this case and others, the observed bands were clear and well defined, suitable for scanning into NIHImage for analysis. NIH-Image produced a graphical representation (Figure 1B) of the gel from which the listed values in Table 1 were derived. The relative molecular weights reported were based on the co-ran molecular weight standards (phosphorylase b, 94 kDa; BSA, 66 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; and trypsin inhibitor, 20 kDa). Fluorescein 1c
and biotin 5c conjugates behaved similarly. Examination of the gels showed that as the hapten to protein coupling ratio increased, so did the apparent molecular weight of conjugates. Gels of the conjugates for coumarin 2c, dansyl 3c, and 3-aminodigoxigenin 4c indicated apparent molecular weights equal to or below that of the BSA standard, with the exception of the most highly substituted 3-aminodigoxigenin conjugate 4c (30:1). No high molecular weight bands corresponding to cross-linked BSA were observed in any sample. MALDI-MS. Spectra were successfully recorded for each of the lyophilized conjugate samples. Each spectrum contained at least two peaks which represented singly (M + H)+ and doubly (M + 2H)2+ charges states (a typical example is shown in Figure 2). The molecular weight of each conjugate was calculated from the peak centroid of the (M + H)+ peak (Table 1). Due to the limited resolution of the MALDI-MS instrument, only broad peaks were observed. Unlike the SDS-PAGE results, the increasing hapten to BSA ratio in each conjugate series was uniformly reflected in the observed molecular weight. LC-ESI-MS. Analysis of conjugates using direct flow injection into the ESI-MS instrument (i.e., no LC) resulted in complicated spectra which could not be deconvoluted. In-line LC separation of the conjugates was required to obtain interpretable data. On the column employed, BSA and modified BSA eluted apart from the low molecular weight impurities. Throughout the chromatography, mass spectra were recorded over two mass ranges, m/z 150-700 and 1200-2400. In the low mass range residual, noncovalently bound impurities in the conjugate sample were detected, while in the high mass range, BSA (MW 66437) and covalently modified BSA were observed. Before purification by dialysis or gel chromatography, we noted the presence of the free hapten (1b-5b) in each conjugate sample (1c-5c, re-
478 Bioconjugate Chem., Vol. 7, No. 4, 1996
Adamczyk et al.
Table 1. Calculated Molecular Weights of BSA Conjugates conjugate molecular weight no. ∆MWa equivb SDS-PAGEc MALDId 1c
359
5
69 732
68 957
15
71 402
71 075
30
73 749
72 274
2c
230
5 15 30
63 729 63 127 63 524
67 037 67 381 67 488
3c
234
5
61 728
67 697
15
63 009
68 921
30
63 193
69 781
5
64 321
68 236
15
66 556
71 416
30
69 843
76 618
5
68 493
68 143
15
69 134
70 618
30
70 345
72 234
4c
5c
473
227
ES/MSe 67 521 (MBSA + 3HAP, 60), 67 870 (MBSA + 4HAP, 100), 68 227 (MBSA + 5HAP, 95), 68 587 (MBSA + 6HAP, 72), 68 944 (MBSA + 7HAP, 20), 69 291 (MBSA + 8HAP, 15) 68 962 (MBSA + 7HAP, 26), 69 306 (MBSA + 8HAP, 53), 69 662 (MBSA + 9HAP, 92), 70 020 (MBSA + 10HAP, 100), 70 381 (MBSA + 11HAP, 92), 70 732 (MBSA + 12HAP, 46), 71 088 (MBSA + 13HAP, 16) 70 379 (MBSA + 11HAP, 10), 70 735 (MBSA + 12HAP, 21), 71 097 (MBSA + 13HAP, 38), 71 452 (MBSA + 14HAP, 72), 71 812 (MBSA + 15HAP, 73), 72 168 (MBSA + 16HAP, 66), 72 524 (MBSA + 17HAP, 100), 72 904 (MBSA + 18HAP, 51) 66 437 (MBSA + 0HAP, 100) 66 437 (MBSA + 0HAP, 100), 66 670 (MBSA + 1HAP, 32), 66 896 (MBSA + 2HAP, 5) 66 437 (MBSA + 0HAP, 100), 66 665 (MBSA + 1HAP, 79), 66 895 (MBSA + 2HAP, 44), 67 120 (MBSA + 3HAP, 10) 66 463 (MBSA + 0HAP, 51), 66 693 (MBSA + 1HAP, 86), 66 932 (MBSA + 2HAP, 100), 67 158 (MBSA + 3HAP, 53), 67 401 (MBSA + 4HAP, 28) 67 396 (MBSA + 4HAP, 1), 67 633 (MBSA + 5HAP, 37), 67 866 (MBSA + 6HAP, 94), 68 106 (MBSA + 7HAP, 100), 68 325 (MBSA + 8HAP, 57), 68 560 (MBSA + 9HAP, 14) 69 280 (MBSA + 11HAP, 17), 69 491 (MBSA + 12HAP, 45), 69 741 (MBSA + 13HAP, 91), 69 962 (MBSA + 14HAP, 100) 66 911 (MBSA + 1HAP, 38), 67 381 (MBSA + 2HAP, 92), 67 852 (MBSA + 3HAP, 100), 68 327 (MBSA + 4HAP, 87), 68 793 (MBSA + 5HAP, 58), 69 264 (MBSA + 6HAP, 36), 69 737 (MBSA + 7HAP, 22) 69 268 (MBSA + 6HAP, 35), 69 737 (MBSA + 7HAP, 73), 70 212 (MBSA + 8HAP, 80), 70 684 (MBSA + 9HAP, 86), 71 153 (MBSA + 10HAP, 100), 71 622 (MBSA + 11HAP, 78), 72 095 (MBSA + 12HAP, 53) 72 100 (MBSA + 12HAP, 28), 72 568 (MBSA + 13HAP, 49), 73 046 (MBSA + 14HAP, 70), 73 519 (MBSA + 15HAP, 63), 73 987 (MBSA + 16HAP, 75), 74 456 (MBSA + 17HAP, 60), 74 933 (MBSA + 18HAP, 83), 75 400 (MBSA + 19HAP, 100) 66 659 (MBSA + 1HAP, 3), 66 891 (MBSA + 2HAP, 29), 67 118 (MBSA + 3HAP, 77), 67 343 (MBSA + 4HAP, 100), 67 569 (MBSA + 5HAP, 93), 67 797 (MBSA + 6HAP, 59), 68 022 (MBSA + 7HAP, 36), 68 249 (MBSA + 8HAP, 19) 68 250 (MBSA + 8HAP, 8), 68 482 (MBSA + 9HAP, 22), 68 702 (MBSA + 10HAP, 47), 68 936 (MBSA + 11HAP, 69), 69 153 (MBSA + 12HAP, 81), 69 379 (MBSA + 13HAP, 93), 69 607 (MBSA + 14HAP, 100), 69 837 (MBSA + 15HAP, 97) 70 526 (MBSA + 18HAP, 15), 70 736 (MBSA + 19HAP, 24), 70 964 (MBSA + 20HAP, 47), 71 195 (MBSA + 21HAP, 77), 71 420 (MBSA + 22HAP, 100), 71 644 (MBSA + 23HAP, 81), 71 872 (MBSA + 24HAP, 83), 72 098 (MBSA + 25HAP, 80)
a Molecular weight increase due to each hapten conjugated to BSA. b Equiv is the molar equivalents of hapten to BSA used in conjugate preparation. c Electrophoresis values are listed as apparent mass determinations. d MALDI values were calculated from the peak centroid. e m/z (M + no. of haptens, relative intensity).
spectively). However, after dialysis or gel chromatography, none of the impurities were observed. In the high mass range, LC-ESI-MS of each conjugate provided a complex spectrum consisting of multiple protein species in multicharged states. The molecular weights of the individual modified protein species in each conjugate were recorded after deconvolution of the data. No significant difference in spectral quality was observed between spectra recorded from the purified or unpurified conjugates. Figure 3 illustrates a typical spectrum for BSA before and after conjugation with activated hapten 5a. The corresponding deconvoluted spectra for the entire 5c conjugate series are shown in Figure 4. No free BSA was detected in the conjugates of series 1c, 4c, and 5c by LC-ESI. Unmodified BSA was the dominant species present in all the conjugates of 2c series and the 3c (5:1) conjugate. Comparison of Methods. Table 2 compares the number of hapten incorporated into BSA as measured by SDS-PAGE, MALDI-MS and LC-ESI. Values for SDS-PAGE and MALDI-MS were determined using the equation:
number of hapten )
(conjugate MW) - (BSA MW) (hapten ∆MW)
The values reported for LC-ESI were calculated using the base peak from the respective spectra listed in Table 1. MALDI-MS, and LC-ESI-MS results indicated hapten density increased in parallel with the hapten to
protein coupling ratio for all but the coumarin conjugate series 2c. SDS-PAGE also showed the same trend for conjugates 1c and 5c, while the data for the other conjugates was in conflict. LC-ESI-MS for conjugate 2c showed that the most abundant species was unmodified BSA at all coupling ratios, while MALDI-MS showed a progression from 2.6 to 4.6 haptens per BSA. LC-ESIMS allowed detection of less abundant species with a hapten density of 1-3. For all the conjugates, LC-ESIMS displayed the range of less prominent individual species of varying hapten density present in each conjugate. DISCUSSION
In light of ever increasing quality and regulatory demands, the characterization of bioconjugates has become critically important. The hapten density of the conjugate is often cited as an important property (25, 26). This is well illustrated in the art of immunogen preparation, where the relationship between the immunoconjugate’s characteristics and the quality of the produced antibody has been debated for some time. The accepted practice is based on the assumption that the highest antibody (IgG) titer is obtained with an hapten density of 15-30 molecules per carrier protein. Higher substitution can result in an IgM response which exceeds that of IgG and produces antibodies of lower affinity. A lower hapten density induces a slower immune response, but often leads to an antibody with higher affinity (25, 26).
ESI−MS of BSA−Hapten Conjugates
Figure 2. MALDI mass spectra of 5c conjugate series.
While great emphasis has been placed on measuring hapten density on bioconjugates, relying solely on hapten density for characterization may be misleading and insufficient. Other factors such as protein cross-linking, coupling with species other than hapten, hapten degradation, and noncovalently bound hapten affect both the measurement of hapten density and the performance of the conjugate. The common method of directly conjugating haptens to proteins with carbodiimides (27) ensures that the cross-linking will occur along with N-acylurea formation on the protein (28-30). Use of pure, isolated activated haptens, or those prepared such that they are free from excess coupling reagent (31), can obviate some of these problems. Still, the methods used to characterize bioconjugates must address these variables before any definitive conclusions can be drawn. Several methods have been used to characterize conjugates in the past (e.g. TNBS titration, UV-difference spectroscopy, gel electrophoresis). Each of these methods have their own limitations (17). Classical biochemical methodologies, however, can be greatly augmented or replaced by bioanalytical mass spectrometry. The two most popular methods for bioconjugate characterization are MALDI-MS and ESI-MS (14). In this paper we evaluated both methods in addition to SDS-PAGE for characterization of five structurally diverse BSA conjugates. The use of pure activated haptens and a high grade of BSA resulted in high yields of soluble conjugates. Solubility of the conjugates allowed for the successful SDSPAGE analysis of each sample. Each sample generated
Bioconjugate Chem., Vol. 7, No. 4, 1996 479
well defined bands. The increase in the apparent molecular weight of the conjugates paralleled the increase hapten to protein coupling ratio in each series. Conjugates of the 2c, 3c, and 4c series gave apparent molecular weights that were below or equal to that of unmodified BSA, but within the series, higher coupling ratios produced higher molecular weights. In these cases SDSPAGE was sensitive to the polarity of the hapten. We concluded that the unmodified protein molecular weight standards were not adequate for assessing the molecular weight of these conjugates. More polar haptens 1a and 5a gave results that paralleled MALDI-MS and ESIMS. Thus, SDS-PAGE does not predictably reflect the degree of hapten incorporation. Its main advantage is in detecting cross-linked conjugates. In earlier work (17), where the conjugation procedure produced conjugates with poor solubility, SDS-PAGE showed smeared bands along with bands for polymerized BSA. An additional drawback to SDS-PAGE is its inherent insensitivity to small changes in molecular weight. The accuracy of SDS-PAGE is poor (
