Profiling of polyclonal antibody light chains by liquid chromatography/electrospray ionization mass spectrometry

Polyclonal antibodies play a critical role in the defense against foreign substances (antigens) in higher vertebrates.1,2 They belong to a group of glycoproteins, which are characterized by high binding affinity and specificity to an antigen. These unique characteristics of antibodies have made them very useful tools for in vitro applications, particularly in the area of clinical chemistry.3 Polyclonal antibodies may be viewed as a composite of monomeric species.4,5 Structurally, an antibody molecule is composed of two heavy chains (∼50 kDa) and two light chains (∼25 kDa) linked by disulfide bonds.6,7 Physico-chemical characterization of polyclonal antibodies presents a substantial challenge due to the complexity of the mixture. Traditional approaches such as gel filtration, ion-exchange chromatography or electrophoresis­do not have sufficient resolution to differentiate among monomeric­species.8-10 Simple, direct methods are needed to determine the unique characteristics of polyclonal antibodies. Mass spectrometry has been demonstrated to be useful for the direct analysis of complex mixtures by profiling.11-15 In this letter we report on the use of liquid chromatography/electrospray ionization mass spectrometry (LC/MS) to document unique patterns (‘profiles’) representing the light chain fragments of four different antigen-specific polyclonal antibodies. Affinity-purified polyclonal antibodies; goat, anti-α-human chorionic gonadotropin (A-hCG); goat, anti-creatine kinase (A-CKMB); and sheep, anti-β-human chorionic gonadotropin (A-MOD) were obtained from the Abbott cell culture facility (Abbott Laboratories, Abbott Park, IL, USA). Affinity-purified rabbit anti-14-3-3 polyclonal antibody (A-14-3-3) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The purity of all studied antibodies was >90% based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All other chemicals were acquired from Sigma Chemical Co. (St. Louis, MO, USA) and were used without further purification. LC/MS was performed using a binary microbore LC system (Microtech Scientific, Sunnyvale, CA, USA) employing a PLRP-S column (8µ, 1000 Å, 50 × 2.1 mm, Polymer Laboratories, Amherst, MA, USA) coupled directly to the mass spectrometer ion source. Mass spectra were acquired using a Sciex API 100 single quadrupole mass spectrometer (Perkin-Elmer-Sciex, Concord, Ontario, Canada) equipped with a heated pneumatically assisted electrospray ion source (Turbo IonSpray). The mass analyzer was calibrated using the manufacturer's protocols and calibration solutions (0.1 mM PPG 1000, 0.2 mM PPG 2000, 0.1% formic acid, and 2 mM ammonium acetate in 1:1 water/methanol). Each antibody sample (5 µL, 3–5 mg/mL in 100 mM NH4HCO3) was reduced to light and heavy chain fragments by reaction with 1 µL of dithiothreitol (DTT, 100 mM) at 60 °C for 10 min. The resulting reaction mixture (1–3 µL) was injected onto the HPLC column and eluted with a 20 min linear gradient of 10:90 to 90:10 acetonitrile/aqueous 0.1% formic acid at a flow rate of 100 µL/min. ESI-MS operating parameters were: interface auxiliary air flow 4–5 L/min at 265 °C and nebulizer (turbo mode) air flow of 1.5 L/min, ionspray voltage of 4720 V, counter electrode (curtain plate) potential of 1000 V, and an orifice potential of 70 V. The instrument scanned a range of m/z 1000–3000 in 0.2 amu steps with a total scan time of 10 s. The resulting spectra were smoothed once using the Kalman method 16 and then deconvoluted to the true mass using software provided by the manufacturer (BioMultiview version 1.2). For this study each polyclonal antibody analyzed was reacted with dithiothreitol (DTT) to reduce the disulfide bonds between heavy and light chains. The resulting mixture of fragments was loaded onto a HPLC column to remove buffer components that interfere with mass analysis (Fig. 1). The ion intensity for light chains was strong and spectra could be recorded without interferences from the heavy chains. Figure 2 is a typical raw spectrum obtained for the light chains and Fig. 3 shows the deconvoluted spectra of the light chain fragments for all polyclonal antibodies studied. The light chain fragments in each sample gave rise to predominant peaks recorded within a molecular mass range between 22 000–23 500 Da. This is consistent with the predicted mass range for light chains based on known sequences from mouse and rabbit monoclonal antibodies. The mass profile recorded from the light chains of each polyclonal antibody was unique to it. Reproducibility of the analyses produced from LC/MS was established by repetitive injections of each sample, which gave indistinguishable chromatograms and mass spectra. Spectra arising from heavy chain fragments were very congested and unresolvable, due to the additional heterogeneity from posttranslational modifications in the C-terminal region (Fc). Total ion current (TIC) chromatogram from LC/MS of DTT reduced A-hCG. Typical raw ESI mass spectrum of A-CKMB light chains. Deconvoluted spectra of light chains of four polyclonal antibodies. Previous reports have demonstrated that the masses of intact IgG monoclonal antibodies and common fragments (Fab, Fc, heavy and light chains) can be determined by ESI-MS.10,17-20 Our initial efforts to analyze intact polyclonal antibodies by LC/MS resulted in congested, unresolvable spectra, due to the complexity of the mixture. Reports by others on the matrix-assisted laser desorption/ionization (MALDI) analysis of polyclonal antibodies resulted in broad peaks giving only the average masses of the polypeptides.21 We chose to use ESI-MS since the polypeptides within polyclonal antibodies have similar molecular masses. Mass measurements with high accuracy (∼0.01%) and resolution are routinely achieved with this technique.22,23 Since the absolute mass accuracy and the resolution of ESI-MS are greater at lower masses, reactions that cleave a protein into smaller fragments are particularly useful. The reduction of the antibody interchain disulfide bonds into light and heavy chain fragments is very specific and easy to perform. This reduction strategy provides a unique advantage by allowing light chain fragments (∼23 kDa) to be readily observed by ESI-MS. Since the light chain fragments rarely contain posttranslational modifications, their mass analysis provides a profile of the various species contained in a polyclonal mixture. As shown in Fig. 3, spectra for the light chain fragments were recorded for each of the studied polyclonal antibodies. In each case the resulting molecular mass profiles for light chains are unique patterns, specific for each polyclonal antibody. In conclusion, we have demonstrated that LC/MS is useful for the profiling of polyclonal antibodies. The resulting spectrum for each of the light chain fragments is a unique ‘fingerprint’ for each polyclonal­sample (Fig. 3). The use of on-line LC/MS allowed for minimal sample manipulation, small sample size, and fast analysis. The procedure described here can be used to compare and monitor various preparations of affinity-purified polyclonal antibodies. Maciej Adamczyk*, John C. Gebler*, Jiang Wu*, * Department of Chemistry (9NM), Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6016, USA

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