Anal. Chem. 1996, 68, 455-462
Automated Analytical System for the Examination of Protein Primary Structure Y. L. Frank Hsieh, HongQi Wang, Chris Elicone, Jonathan Mark, Stephen A. Martin,* and Fred Regnier
PerSeptive Biosystems Inc., 500 Old Connecticut Path, Framingham, Massachusetts 01701
This paper describes an automated analytical system for the examination of protein primary structure in which (i) the target protein is first purified by immunoaffinity chromatography, (ii) subsequent chromatographic and chemical reaction steps in the sequencing process are directly coupled, (iii) buffer exchange between these unit operations is achieved while the protein is absorbed on a mixed bed of strong ion exchange sorbents, (iv) proteolysis occurs in an immobilized trypsin column having a 101000 fold-excess of enzyme, (v) the tryptic digest is directly transferred to a perfusion dilute capture column where it is concentrated and rapidly desalted, and (vi) peptides eluted from the dilute capture column and analytical microbore and capillary perfusion reversedphase chromatography columns are analyzed by either single-stage mass spectrometry (MS) or tandem MS/MS. Protein structure variants were easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb Ao was verified. Sequence analysis has become an integral part of the production and quality control of human therapeutic proteins in the biotechnology industry. During biosynthesis and purification, structure variants may be produced through genetic drift, biosynthetic errors in transcription or translation, or enzymatic degradation during fermentation or cell lysis, or the protein may be chemically modified in the early stages of downstream processing. The presence of primary structure variants is of great concern because they may be of diminished biological activity or immunogenic. Immunogenicity indicates that the immune system (i) has targeted the substance as foreign, (ii) produced antibodies that are directed against some portion of the structure, (iii) will sequester the therapeutic protein on subsequent use, and (iv) may modulate or reduce the therapeutic efficacy of the protein in chronic use. Recently, peptide mapping coupled with enzymatic deglycosylation and mass spectrometry has become the method of choice for examining posttranslational modifications.1 Ideally, primary structure analyses would be carried out during biosynthesis, after cell lysis (when it applies), and after harsh steps in the purification process. This would allow process failures to be both detected and localized. The fact that the target protein is present in a complex biological matrix and all known methods of primary structure analysis require purified protein presents a challenging problem. The protein must first be purified by multiple chromatographic or electrophoretic operations. Subsequent to purification, in most cases it must (i) be reduced and (1) (a) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183196. (b) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877-884. 0003-2700/96/0368-0455$12.00/0
© 1996 American Chemical Society
alkylated, (ii) undergo several desalting or buffer exchange steps, (iii) be digested with a proteolytic enzyme, (iv) dilute capture of the proteolytic peptides, (v) have peptide fragments separated by reversed-phase chromatography, and (vi) in many cases be either sequenced by the Edman method or subjected to mass spectral analysis. Other than liquid chromatography-mass spectrometry (LC-MS), all the steps in this process are executed manually, often requiring several days to complete the analysis, even in the best case. Furthermore, multiple manual manipulations of small samples characteristically give poor sample recovery, increase the probability of contamination, are slow, and make it more difficult to analyze large numbers of samples. A major objective of this research was to eliminate these manual transfers; i.e., once the sample was introduced into the system, it would be automatically carried through all the steps in the analysis to detection. This paper examines a new method in which the target protein is sequentially (i) purified by immunoaffinity chromatography, (ii) desalted, buffer exchanged on a mixed-bed strong ion exchange sorbent, and (iii) digested in an immobilized trypsin bed;2 then, (iv) the trypsin digest is fractionated and captured by a short perfusion capillary RPC column, (v) the trypsin digest is separated by analytical reversed-phase chromatography, and (vi) the separated peptides are analyzed by electrospray ionization mass spectrometry (ESI-MS). Human hemoglobin (Hb) Ao and the variant Hb S associated with sickle-cell syndrome, both spiked with several human plasma proteins, were chosen as the model proteins for this study. Hb S is the result of a genetic mutation which results in the substitution of valine for glutamic acid at position 122 in the β-chain of Hb Ao. These protein mixtures provide an ideal system to test a new system for primary structure analysis because (i) the structures of both Hb Ao and Hb S are known, (ii) they vary in structure at a single site, (iii) their tryptic maps are relatively complicated, (iv) the mixtures contain a variety of non-hemoglobin plasma proteins and (v) samples are easily obtained. A variety of ways have been developed to determine the position of amino acid substitution in hemoglobin variants, including MS alone,3-12 LCMS,13-17 and LC-MS/MS. MATERIALS AND METHODS Chemicals. Human normal-cell hemoglobin (Hb Ao) (R-chain MW ) 15 126.4; β-chain MW ) 15 867.3) and sickle-cell hemoglobin (Hb S) (R-chain MW ) 15 126.4; β-chain MW ) 15 837.3) (2) (a) Kassel, D. B.; Consler, T. G.; Shalaby, M.; Sekhri, P.; Gordon, N.; Nadler, T. In Techniques in Protein Chemistry VI; Crabb, J. W., Ed.; Academic Press: 1995; pp 39-46. (b) Hsieh, F. Y. L.; Verentchikov, A.; Elicone, C.; Hines, W.; Wang, H.; Vestal, M.; Regnier, F.; Martin, S. 11th Montreux Symposium on Liquid Chromatography Mass Spectrometry (LC/MS; SFC/ MS; CE/MS; MS/MS), Switzerland, November 1994; p 49.
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with ∼50% nonionic stabilizers, human globulins, human fibrinogen, human apo-transferrin, human serum albumin, and human thrombin were purchased from Sigma Chemical Co. (St. Louis, MO). Affinity purified goat anti-human hemoglobin (whole) (3.3 mg/mL) was purchased from Cortex Biochem (San Leandro, CA). Hemoglobin Ao and S were mixed, respectively, with several different human plasma proteins (human globulins, human fibrinogen, human apo-transferrin, human serum albumin, human thrombin, 2 mg/mL each) as samples. Trypsin was purchased from Worthington Biochemical Co. (Freehold, NJ). Liquid Chromatography. Chromatographic separations and enzyme reactions were carried out on an INTEGRAL microanalytical workstation (PerSeptive Biosystems, Cambridge, MA) equipped with an autosampler and three computer-controlled, 10port biocompatible valves. When column effluents were monitored by an absorbance detector, the system detector was modified by the substitution of a high-pressure microbore flow cell (Thermo Separation Products, Fremont, CA). Five columns were plumbed into the valve system of the INTEGRAL (Figure 1A). This plumbing configuration allows columns to be equilibrated, loaded, concentrated, and eluted separately or in series as described below. Solvents used in the system were as follows: 1A, 0.1% triflouroacetic acid (Pierce, Rockford, IL); 1B, HPLC grade water (J.T. Baker, Phillipsburg, NJ); 1C, 10 mM ammonium acetate, pH 7.5 (Sigma); 2A, 80% acetonitrile (J.T. Baker) with 0.1% triflouroacetic acid; 2B, 12 mM hydrochloric acid (J.T. Baker) with 1.5% acetonitrile; and 2C, 100 mM Tris-HCl (Sigma) with 10 mM calcium chloride, pH 8.0 (J.T. Baker) and 2 M sodium chloride (Sigma). A 1- × 250-mm Vydac C18 (The Separations Group, Hesperia, CA) analytical reversed-phase chromatography column was used in these studies. A Poros reversed-phase column (R3), 508 µm × 50 mm, was employed for dilute capture (PerSeptive Biosystems). Automation of the Five-Column System for the Analysis of Hemoglobin. The overall system layout for the five columns coupled to an ESI-MS system is shown in Figure 1A (Figure 1A is shown with all valves in their clockwise position). The dilute (3) Van Emon, J. M.; Lopez-Avila V. Anal. Chem. 1992, 64, 79A-86A. (4) (a) Li, Y. L.; Hsieh, Y. L.; Henion, J. D.; Ganem, B. J. Am. Soc. Mass Spectrom. 1993, 4, 631-637. (b) Norregaard Jensen, O.; Roepstorff, P.; Rozynov, B.; Horanyi, M.; Szelenyi, J.; Hollan, S. R.; Aseeva, E. A.; Spivak, V. A. Biol. Mass Spectrom. 1991, 20, 579-584. (c) Shackleton, C. H. L.; Falick, A. M.; Green, B. N.; Witkowska, H. E. J. Chromatogr. 1991, 562, 175-190. (5) Martin de Llano, J. J.; Schneewind, O.; Stetler, G.; Manning, J. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 918-922. (6) Wada, Y.; Matsuo, T.; Sakurai, T. Mass Spectrom. Rev. 1989, 8, 379-434. (7) Martin de Llano, J. J.; Jones, W.; Schneider, K.; Chait, B. T.; Manning, J. M.; Rodgers, G.; Benjamin, L. J.; Weksler, B. J. Biol. Chem. 1993, 268, 27004-27011. (8) Springer, D. L.; Bull, R. J.; Goheen, S. C.; Sylvester, D. M.; Edmonds, C. G. J. Toxicol. Environ. Health. 1993, 40, 161-176. (9) Glamsta, E. L.; Nyberg, F.; Silberring, J. Rapid Commun. Mass Spectrom. 1992, 6, 777-780. (10) Falick, A. M.; Shackleton, C. H.; Green, B. N.; Witkowska, H. E. Rapid Commun. Mass Spectrom. 1990, 4, 393-400. (11) Prome, D.; Blouquit, Y.; Ponthus, C.; Prome, J. C.; Rosa, J. J. Biol. Chem. 1991, 266, 13050-13054. (12) De Biasi, R.; Spiteri, D.; Caldora, M.; Iodice, R.; Pucci, P., Malorni, A.; Ferranti, P.; Marino, G. Hemoglobin 1988, 12, 323-336. (13) Covey, T. R.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63, 11931200. (14) Piot, J. M.; Zhao, Q.; Guillochon, D.; Ricart, G.; Thomas, D. FEBS Lett. 1992, 299, 75-79. (15) Jensen, O. N.; Roepstorff, P. Hemoglobin 1991, 15, 497-507. (16) Stachowiak, K.; Dyckes, D. F. Pept. Res. 1989, 2, 267. (17) Ingram, V. M. Nature 1957, 180, 326-328.
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capture and analytical reversed-phase columns were equilibrated in solvent 1A at 50 µL/min for 5 min prior to use. The mobilephase solvents can be purged at any of the LC processing steps with the following settings (Figure 1A): injector value, load position; valve 1, counterclockwise, positions 1-2, 3-4, etc. connected; and valve 2, clockwise, positions 1-10, 9-8, etc. connected. The immobilized trypsin column (Porozyme, PerSeptive Biosystems) and the anti-hemoglobin immunodetection (ID) cartridge (PerSeptive Biosystems) are then sequentially equilibrated in 100% 2C and 100% 1C, respectively. Step 1. The sample is loaded through the autosampler onto the ID cartridge and washed with solvent 1C at 300 µL/min for 5 min (Figure 1B). The immobilized anti-hemoglobin antibody binds specifically with hemoglobins, and unbound proteins are eluted to waste-2 (valve 1). Step 2. The ID and the POROS HS 2.1 -× 30-mm strong cation exchanger cartridge (PerSeptive Biosystems) are equilibrated in solvent 1C. The ID cartridge is then eluted at 500 µL/min with solvent 2B onto the cation exchanger. Both columns are then washed with solvent 1C to bring the pH value up to 7.5 in the buffer exchange column (Figure 1C). In subsequent experimental setups, a mixed (cation, anion)-bed exchange column replaced the strong cation exchange column. This increased the range of proteins retained during the wash step with solvent 1C (see Results and Discussion). Step 3a. The inject valve is switched to remove the ID cartridge from the flow and enable the syringe pump in the INTEGRAL to inject 100 µL of 2 M NaCl onto the cation exchange column to release the bound protein (Figure 1D). Step 3b. The cation exchange column, the Poroszymeimmobilized trypsin cartridge (PerSeptive Biosystems), and a 508µm × 50-mm R3 dilute capture reversed-phase (RP) column (PerSeptive Biosystems) are all brought in line at a flow of 50 µL/min of solvent 2C. The hemoglobin antigen is released from the cation exchanger and flows through the trypsin cartridge, where it is digested, and the resulting peptides are retained by the dilute capture column (Figure 1D). Step 4a. Solvents 1A and 2A are used for purging the lines. Step 4b. The salts are then rapidly washed from the dilute capture column to waste-3 (valve1) with solvent 1A for 1 min (Figure 1E). Step 5. Tryptic peptides from hemoglobin captured on the capillary perfusion RP column are then separated by this column and the analytical RP column (the analytical RP column is always connected to the electrospray interface for robotic analysis). The RP column is eluted with a gradient of 1%/min solvent 2A for 50 min (Vydac C18 RP column) at a flow rate of 50 µL/min (Figure 1F). In the automated mode employed for this five-column chemistry, the INTEGRAL controls the start time of the ESI-MS, initializing data acquisition. Liquid Chromatography-Mass Spectrometry (LC-MS). A PE-Sciex API III plus atmospheric pressure ionization (API) triple quadrupole mass spectrometer (Thornhill, ON, Canada) with an upper mass limit of m/z 2400 was used. During the on-line ion spray LC-MS acquisition, the step size is 0.5 amu, with a 5.18 s scan rate (dwell time, 2.3 ms) from m/z 400 to 1500 at declustering potentials of 60 V. The total effluent of 50 µL/min was directed unsplit to the articulated ion spray LC-MS interface. The sprayer was positioned ∼1 cm off-axis and 1 cm away from the ion sampling orifice and maintained at 5 kV with 0.8 L/min
Figure 1. Five-column chromatography system for the isolation and characterization of human hemoglobins. (A) Overall plumbing system layout for the automatic multidimensional LC-MS (immunoaffinity f buffer exchange f enzyme f dilute capture f RP chromatography f MS) process. (B) Step 1, immobilized antibodies (ID cartridge) bind specific antigens. (C) Step 2, bound antigens are desorbed by 12 mM HCl from the ID cartridge to a perfusion buffer exchange column. The pH of the antigens is brought up to 7.5. (D) Step 3a, 2M NaCl is introduced into the ion exchange column. Step 3b, antigens are eluted out from ion exchange column, flow through the immobilized perfusion trypsin column, and are captured by the capture and desalt capillary perfusion RP chromatography column. (E) Step 4a, lines are purged with 0.1% TFA. Step 4b, salts are rapidly removed from the capillary perfusion RP chromatography column prior to separation and mass spectrometric analysis. (F) Step 5, tryptic peptides are separated from the capillary perfusion RP chromatography column and the C18 analytical column to mass spectrometry.
nebulizing nitrogen and 1.0 L/min curtain nitrogen gas. Poly(propylene glycol) in 80:20 CH3CN/H2O (5 mM NH4OAc) was continuously introduced at 2 µL/min at ambient temperature by an infusion pump (Harvard Apparatus, South Natick, MA) and used for tuning and mass axis calibration for each mass-resolving quadrupole (Q1 and Q3) at unit resolution with 2 ms dwell time and 10 scans summed at a step size of 0.1 amu. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Argon collision gas was introduced into the collision cell (Q2) for the LC-MS/MS experiments with a collision gas thickness of 180 × 1012 atoms/cm2, a collision energy of 45 eV (laboratory frame), and declustering energy of 60 V. Product ions were acquired at 0.5 amu step size with 2.5 ms dwell time from m/z 50 to 1000. Antibody Immobilization. This antibody was immobilized on an XL-type ID cartridge18 from PerSeptive Biosystems, Inc. This cartridge comes in the form of a kit with an automated immobilization procedure which was performed on the LC instru-
ment. This procedure was used for the preparation of all immunosorbents described in this paper. In this immobilization, antibody is first captured by protein G bonded to the XL support and then covalently bound to the support by cross-linking the antibody to protein G with dimethyl pimelimidate (DMP). Unreacted DMP was then quenched with ethanolamine. The binding capacity of the cartridge was determined with human hemoglobin Ao (2 mg/mL in 10 mM PBS) and found to be ∼80 µg on a 2.1× 30-mm column, i.e., 2 mg/mL. Immunosorbent was packed in a 2.1- × 30-mm PEEK column (Upchurch, Seattle, WA) by highpressure slurry packing in 0.1 M Tris buffer (pH 8). Antigen elution was achieved with 12 mM HCl. RESULTS AND DISCUSSION General System Design. Automation in analytical chemistry is frequently associated with robotics. After detailed analysis, it was concluded that a robotic system would not provide a suitable solution for the following reasons. The limitations of the robotic Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
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Figure 2. Lifetime binding affinity of HSA to XL anti-HSA cartridges (antibody is immobilized to protein G).
approach are due more to (i) the multiple pieces of apparatus used in the analysis, (ii) the multiple syringes or pipets being used to transfer anogram quantities of analytes, (iii) the difficulty in removing all the sample from microvessels such as fraction collector tubes and the proteolytic digestion flask, and (iv) contamination from the open nature of the apparatus. It was concluded that a multivalved apparatus which allows automated, directed transfer of analyte between the various unit operations in the process provides a better solution. This conclusion was based on the following facts: (i) direct coupling of the various unit operations by valves and transfer lines eliminates the need for syringe or pipet transfers, (ii) the internal volume of a valve and tubing connecting tandem chromatography columns or a chromatography column and an immobilized enzyme reactor can be
