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Anal. Chem. 1991. 63.732-739
in the liquified SF6that exists in pressurized cylinders. The results of analyzing SF6 stored as a liquid suggest that significant improvements might be achieved in maintaining sample stability by compressing reference samples into the liquid phase. ACKNOWLEDGMENT We are grateful to M. C. Siddagangappa of the Indian Institute of Science, Bangalor, India, who provided valuable assistance in the early stages of this work, and to Daryl DesMarteau, Clemson University, for providing us with the S2FIoused in this work. LITERATURE CITED Greenberg, L. A.; Lester, D. Arch. Ind. Hyg. Occup. M e d . 1950, 2 , 350-353. sunders, J. P.; Shashka, M. M.; Decarlo, M. R.; Brown, E. Arch. Ind. Hyg. OCCUP. Med. 1953, 8 , 436-445. Griffin, G. D.; Easterly, C. E.; Sauers, I.; Ellis, H. W.; Christophorou, L. G. Toxicol. Enwiron. Chem. 1984, 9 , 139-166. Kurka, K.; Morris, M. D.; Votaw, Griffin, G. D.; Nolan, M. G.;Sauers, I.; P. C. In Vitro 1989, 2 5 , 673-675. American Conference of Governmental and Industrial Hygienists, 5th ed.; American Conference of Government 8 Industrial Hygienists Association: Cincinnati, OH, 1986. Votaw, P. C.; Griffin, G.D. J. Phys. D : Appl. Phys. 1988, Sauers, I.; 2 1 , 1236-1238. Sauers, I.; Siddagangappa, M. C.; Harman, G.; Van Brunt, R. J.; Herron, J. T., Proceedings of the 6th International Symposium on High Voltage Engineering, New Orleans, LA, 1989; Vol. 1, pp 2508-2511. Herron, J. T. I€€€ Trans. €le&. Insul. 1987, € I - 2 2 , 523-525. Van Brunt, R . J.; Herron, J. T. I€€€ Trans. Electr. Insul. 1990, € I 2 5 , 75-94. Federal Register, Air Contaminants Final Rule, OSHA, Jan 19, 1989. Janssen, F. J. J. G. Kema Sci. Techno/. Rep. 1984, 2 , 9-18. Janssen, F . J. J. G. Proceedings of the 5th International Symposium on Gaseous Dielectrics, Gaseous Dielectrics V : Pergamon Press: New York, 1986; pp 153-162.
(13) Sauers, I.; Harman, G.; Olthoff, J. K.; Van Brunt, R. J. I n Procdings of the 6th International Symposium on Gaseous Dielectrics , Geseous Dielectrics V I ; Christophorou, L. G., Sauers, I.,Eds.; Plenum Press: New York, 1991; pp 528-537. (14) Cohen, B.; Mac Diarmid, A. G. Inorg. Chem. 1982, 1 , 754-756. (15) Olthoff, J. K.; Van Brunt, R . J.; Sauers, I.J. Phys. D : Appl. Phys. 1989. 2 2 , 1399-1401. (16) Farber, M.; Harris, S. P.; Cooper, D. E.; Khazei, M. J . Phys. D : Appl. Phys. 1989, 2 2 , 233-234. (17) Hanrahan. J. M.; Patterson, A. R. J . Chromatogr. 1980, 193, 265-275. -..- .
(18) Van Brunt, R. J. J . Res. Nat. Bur. Stand. 1985, 9 0 , 229-253. (19) Dibeler, V. H.; Mohler. F. L. J . Res. Nat. Bur. Stand. 1948, 4 0 , 25-33. (20) Cornu, A.; Massot, R. Compihtion of Mass Spectral Data, 2nd ed.; Heyden: New York, 1975; Vol. 1, p 77A. (21) Trost, W. R.; McIntosh, R. L. Can. J . Chem. 1952, 2 9 , 508-525. (22) Benson, S. W.; Bott, J. Int. J . Chem. Kinet. 1969, 1 , 451-458. (23) Herron, J. T. Int. J . Chem. Kinet. 1987, 19, 129-142. (24) Tait, J. C.; Howard, J. A. Can. J. Chem. 1975, 5 3 , 2361-2362. (25) Van-Brunt, R. J.; Sauers, I.J . Chem. Phys. 1986, 85, 4377-4380. (26) Vorbev, M. D.; Filatov, A. S.; Englin, M. A. J. Gen. Chem. USSR (Engl. Trans/.) 1975, 4 4 , 2677-2679. (27) Roberts, H. L. J . Chem. SOC. 1962, 3183-3185. (28) S i b o t t o m , H. W.; Tedder, J. M.; Watton, J. C. Trans. Faraday SOC. 1969, 65, 2103-2109. (29) Cohen. 8.; MacDiarmid, A. G. Inorg. Chem. 1965, 4 , 1782-1765. Harman, G. (30) Olthoff, J. K.; Van Brunt, R. J.; Herron, J. T.; Sauers, I.; Conference Record of the 1990 IEEE International Symposium on €lectrical Insulation; IEEE: New York, 1990; pp 248-252.
RECEIVED for review August 16, 1990. Accepted January 7, 1991. The identification of commercial materials and their sources is made to describe the experiment adequately. In no case does this identification imply recommendation by the National Institute of Standards and Technology nor does it imply that the product is the best available. This work was supported by the Office of Energy Storage and Distribution, Electric Energy Systems Program, U.S. Department of Energy.
Packed-Capillary Liquid Chromatography/Ion-Spray Tandem Mass Spectrometry Determination of Biomolecules Eric C. Huang' and Jack D. Henion*
Drug Testing and Toxicology, Cornell University, 925 Warren Drive, Ithaca, New York 14850
Coupling packed-caplllary liquid chromatography (HPLC) wlth a tandem quadrupole mass spectrometer (MS/MS) system capable of sampling Ions formed at atmospheric pressure via the pneumatically asslsted electrospray (Ion spray) LC/MS Interface Is demonstrated. The low flow rate required for performing packed-capillary LC separatlon can be achieved with minimum modlflcation of a commerclally avallabie micro LC pump. Precoiumn flow splitting Is successfully lmpiemented for gradient separation in peptlde-mapping applicatlons with a column Internal dlameter of 320 pm. Peptide standards, protein tryptic digests, and a crude peptkle mixture from Industry are used to demonstrate the feasibility for utilizing the packed-capillary LC/MS system in a variety of applications. High-sensitlvlty micro LC/MS peptide mapping and molecular welght determination can be performed routinely via ion-spray LCIMS wRh Injected sample amounts as low as 10 pmol. Peptide sequence lnformatlon Is obtained via on-line packed-capillary LC/MS/MS.
* To whom
correspondence should be addressed. Current address: M e r c k Sharp & Dohme Research Laboratories, West Point, PA 19486.
INTRODUCTION Mass spectrometry exhibits a variety of unique capabilities for a broad range of applications and has enjoyed increased use for the analysis of complex mixtures. The major extension of the applicability of mass spectrometry in biomedical research has in large measure been attributable to the development of new ionization techniques such as fast atom bombardment ( I ) , plasma desorption (2),laser desorption (3)and their associated sample-handling methods. More recently, matrix-assisted laser desorption ionization ( 4 ) and ion evaporation ionization via the electrospray method (5)have been developed (or, more accurately, improved) and demonstrated as alternative soft ionization methods. Their use in the application to biomedical mass spectrometry research has grown rapidly in the last few years and in some cases they have shown superior performance to other soft ionization methods. In addition, a mass spectrometer in tandem configuration can provide a method for mixture analysis without preseparation of components or sample cleanup procedures. However, it is also known that sample cleanup or prefractionation of a mixture Prior to mass analysis is Vital for analyzing redworld biologically important mixtures.
0003-2700/91/0363-0732$02.50/00 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
High-performance liquid chromatography (HPLC) is one of the most widely used methods for separating mixtures of biomolecules such as proteins and peptides. The method has been practiced over the last two decades and has become widely accepted. The combination of the full capabilities of HPLC and mass spectrometry in a tandem format is extremely attractive for the characterization of proteins and peptides in complex mixtures. Due to the fundamental differences in operating conditions, coupling HPLC with a mass spectrometer equipped with a conventional ion source has traditionally been a difficult task. Nevertheless, important progress in the field has been achieved during the last decade. This includes the development of moving-belt (6),thermospray (3, particle beam (8),and continuous-flow FAB LC/MS interfaces (9). While these interfaces provide a variety of ways for performing on-line LC/MS, their use for characterizing large biomolecules has shown limited success due largely to the difficulty of forming gas-phase molecular ions from the condensed phase. Recent advances in atmospheric pressure ionization mass spectrometry (APIMS) in conjunction with refinements of ion evaporation ionization through the electrospray technique have expanded the capabilities of LC/MS. Using this ionization technique, biomolecules as large as 130000 Da have been successfully ionized, mass-analyzed, and detected (IO). Since the ion formation process for this technique is based upon ion evaporation ionization and is described as being field-assisted desorption of solute ions from rapidly evaporated liquid droplets (11-14), the optimum condensedphase flow for the pure electrospray method is less than 10 pL/min (25). Higher condensed-phase flow under pure electrospray conditions results in a decrease in the ionization efficiency and an unstable liquid droplet formation process. Thus, a postcolumn split is necessary for coupling conventional HPLC with APIMS coupled to a pure electrospray interface unless a packed-capillary column with an internal diameter of less than 500 pm is used to perform the LC separation (16, 17).
Problems associated with the limited acceptable flow range for electrospray LC/MS may be circumvented by implementing the pneumatically assisted electrospray (ion spray) technique (28). This approach improves droplet formation and stabilizes the ion current generated over a broad range of liquid flow rates and eluent compositions. This allows the use of mobile phases containing solvent with high surface tension such as those having a high aqueous content. In our laboratory high-sensitivity microbore LC/MS with a mobile phase flow of 40 pL/min is routinely performed by using the ion-spray interface without the need for a postcolumn split (18,19). In fact, liquid flow as high as 200 pL/min has been used via this interface without a postcolumn split (20). However, there are circumstances (e.g., to accommodate the need torecover a large portion of the sample for other uses and/or to achieve high-sensitivity LC/MS provided by using the packed-capillary column) in which the ion-spray interface may be operated under lower liquid -flow conditions. HPLC separations with the use of packed-capillary columns require very low volumetric flow. This requirement fulfills the condition of potential high-sensitivity performance with the ion-spray LC/MS interface. Hence, the use of packedcapillary HPLC and its coupling to APIMS via the ion-spray interface is the focus of this report. We describe a packedcapillary (i.e., columns of 500 pm X 100 mm and 320 pm X 50 mm) LC/MS system that can provide linear gradients for simultaneous high-resolution peptide separation and molecular weight determination. The capability for gaining structural information by LC/MS/MS is also demonstrated. Peptide standards and real-world peptide mixtures, including protein tryptic digests and crude synthetic peptide mixtures, are used
733
Figure 1. Schematic diagram of the packed-capillary LC system: (A) dual-syringe micro LC pump; (B) static mixer: (C) precolumn flow split tee; (D) fused-silica capillary flow split line; (E) injector; (F) packedcapillary LC column; (G) fused-silica transfer line.
for demonstrating the feasibility of this packed-capillary LC/MS system for solving biological problems. EXPERIMENTAL SECTION Chemicals. The peptide standards and proteins used in this work, e.g., fibrinopeptide A, substance P, bradykinin, human serum albumin, bovine cytochrome C, and bovine dephosphorylated &casein were all purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. The enzyme used to digest the proteins was trypsin treated with L-[1-(tosylamido)-2-phenylethyl]chloromethylketone (TPCK) and was obtained from Sigma Chemical Co. The crude synthetic peptide mixture was provided by the Eastman Kodak Co. (Rochester, NY). HPLC grade acetonitrile was obtained from J. T. Baker Inc. (Phillipsburg, NJ), and HPLC grade water was obtained from Fisher Scientific (Rochester, NY). High-purity trifluoroacetic acid (TFA) and ammonium bicarbonate were also purchased from Sigma Chemical Co. Enzymatic Digestion. Bovine cytochrome C (1 mg, approximately 76 nmol) and bovine dephosphorylated P-casein (1 mg, approximately 42 nmol) were digested with TPCK-treated trypsin for 24 h in a 37 "C water bath with a substrate-to-enzyme ratio of 50:l (w/w) in 50 mM ammonium bicarbonate buffer solution with its pH adjusted by 1 M ammonium hydroxide to a value of pH 8.5. The digested solution was then lyophilized and redissolved in water containing 0.05% TFA. Packed-Capillary Columns. Two different packed-capillary LC columns were used in this study. The first was a 500 pm internal diameter, 100 mm long column packed with 5-pm LC-308 packing material (bonded with c8 stationary phase, 300-8, pore size) generously provided by Supelco, Inc. (Bellefonte,PA). The column was encased in a 10 cm long stainless steel tube to facilitate handling. Universal fiigertight fittings (Upchurch Scientific, Inc., Oak Harbor, WA) were used at both column inlet and outlet for direct connection to the injector and detector. The second column was 320 pm internal diameter, 50 mm long packed with 3-pm particles of 80-8, pore size and bonded with c18 stationary phase generously provided by LC Packing International (San Francisco, CA). This column was made of fused-silica capillary tubing and protected by a piece of 1/16 in. i.d. stainless steel tubing. The column terminates with a porous microfrit and connects with a fused-silica outlet capillary (50 pm i.d. X 70 mm) to facilitate connection to the detector. Liquid Chromatography System. A schematic diagram of the packed-capillary HPLC system used in this study is shown in Figure 1. An Applied Biosystems Inc. (Foster City, CA) Model 140A dual-syringe micro LC pump (A) was used to deliver solvent gradients; the liquid flow was bypassed from the original 200-pL dynamic mixer to a Brownlee 1-mm X 30-mm guard cartridge (Applied Biosystems Inc., Foster City, CA) which served as a static mixer (B) with a mixing volume of 52 pL. A high-pressure mixing tee obtained from Upchurch Scientific, Inc. (Oak Harbor, WA) (C) with 3.1-pL dead-volume was inserted between the static mixer and sample injector (E) to act as the precolumn flow splitting device. The split line (D, 50 pm X 550-650 mm, used here only with the 320-pm packed-capillary column) was a piece of fusedsilica capillary tubing (Polymicro Technologies, Phoenix, AZ). Its internal diameter and length were predetermined to accommodate the desired split ratio and vary with certain applications. The
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
packed-capillary column (F) was connected directly to the sample injector, while particular effort was made to avoid additional extracolumn dead volume. For the 500-pm X 100-mmC8column, a Rheodyne (Cotati, CA) Model 9125 biocompatible sample injector with either a 5- or IO-pL external loop was used. For the 320-wm X 50-mm C18column, a Model 7520 syringe-loadingsample injector with an internal sample disk of I-pL rotor volume was used. The column outlet was connected to the detector via a fused-silica capillary transfer line (G) to minimize extracolumn band broadening due to the dead volume introduced here (21). Two detectors were used in this experiment: a Kratos Model 757 UV detector (Applied Biosystems Inc., Foster City, CA) and a Sciex TAGA 6000E atmospheric pressure ionization (API) triple-quadrupole mass spectrometer (Thornhill, Ontario, Canada). Details of the mass spectrometer are given in the next section. The UV flow cell used here was originally developed for capillary electrophoresis by Applied Biosystem Inc. It consists of a small section of 0.05 mm id., 0.36 mm 0.d. fused-silica capillary stripped of its polyimide coating, which is secured and alligned in a focusing support. The UV detector was set at a wavelength of 214 nm for detection of the peptide carbonyl bonds. The mobile phase used for all the packed-capillary HPLC separations in this study consisted of 0.15% TFA/water as solvent A and 0.15% TFA/ acetonitrile as solvent B. Solvent degassing was performed by purging (ca. 15 min) with ultrahigh-purity grade helium (Linde Specialty Gases, Danbury, CT). Mass Spectrometry. A SCIEX (Thornhill, Ontario, Canada) TAGA 6000E triple-quadrupole mass spectrometer equipped with an atmospheric pressure ionization (API) source was used with a mass range of 1400 Da. The interface for introducing the HPLC effluent to the APIMS was a pneumatically assisted electrospray (ion spray) interface that was constructed in-house. Details of the interface construction have been described previously (18). The sprayer may be floated at *3 kV or higher, the polarity depending upon the operational mode (Le., positive- or negative-ion detection) of the mass spectrometer. In the present study the mass spectrometer was operated in the positive-ion mode. Ions generated from the ion-spray interface via the ion evaporation mechanism were sampled into the mass spectrometer by a potential difference between the sprayer and the sampling orifice. The sampling orifice was a 100 pm diameter hole at the end of a conical skimmer. A flow of ultrapure nitrogen (Linde Specialty Gases, Danbury, CT) bathed the atmospheric pressure side of the orifice to minimize solvent clusters and preclude particulate matter from entering the mass spectrometer. For LC/MS operation, the first quadrupole was scanned from 300 to 1400 Da with unit mass resolution (based on 20% valley definition) at a scan rate of 7 s/scan. For LC/MS/MS experiments, the parent ions were selected and focused by the first quadrupole (Q-1)for transmission into the RF-only quadrupole (i.e., collision cell, Q-2) where the parent ion was subjected to collision-induced dissociation. The collision gas thickness was preset at 200 X IO1*atom/cm*, which corresponded to a system Torr. Depending on the experipressure reading of 2.2 X ments, the collision energy was controlled by the offset voltage of the RF-only quadrupole. The LC/MS/MS daughter ion mass spectra were acquired by scanning the third quadrupole (Q-3)from m / t 10 to 1400 with unit mass resolution (20%valley definition) at a scan rate of 10 s/scan.
RESULTS AND DISCUSSION Our previous work has shown that the ion-spray LC/MS interface may be used for coupling various types of separation systems including supercritical fluid chromatography (SFC), microbore liquid chromatography, ion chromatography (IC), and capillary electrophoresis (CE)to APIMS (22). The remaining discussion is confined to packed-capillary HPLC and its coupling with APIMS. Gradient Formation. The unique properties of packedcapillary HPLC and its coupling to MS provide several advantages over conventional HPLC. For example, low solvent consumption dramatically reduces solvent waste, and improved mass sensitivity enhances overall sensitivity for LC/MS. However, there are many practical difficulties involved with using packed-capillary HPLC, so the technique
is not yet widely used for problem-solving. The major problems associated with the use of packed-capillary HPLC for problem-solving are the difficulties involved with generating a reliable, reproducible solvent gradient at a flow of less than 10 pL/min. This is especially true when one deals with protein and/or peptide mixtures where a solvent gradient is essential for separating such complex samples. To overcome this obstacle, we have investigated two different approaches. The first approach was to reduce the total tubing volume between the dual-syringe pump and the sample injector by using small internal diameter tubing and a mixing device with very small mixing volume. When the split device (C and D in Figure 1)was removed from the HPLC system, a stable and reproducible linear solvent gradient (95% aqueous to 5% aqueous) could be generated at a liquid flow as low as 10 pL/min. For flow lower than 10 pL/min, the gradient performance from this approach was not acceptable mainly due to inadequate reproducibility. Although the high accuracy of a solvent gradient is desirable, a reproducible gradient flow is more important when the technique is applied for problem solving on a daily basis. This setup (i.e., no precolumn flow splitting) was used for all the separations involving 500 pm i.d. packed-capillary column. In general, when columns with reduced internal diameter for HPLC separations are used, the volumetric flow must be scaled down in proportion to the square of the column intemal diameter (23). Packed-capillary columns with an internal diameter of 320 pm require a flow of 3-5 pL/min for optimum separation efficiency. This low-flow requirement imposes a very difficult challenge for generating a solvent gradient. Novotny et al. have developed a “step gradient” (24)technique to allow reversed-phase HPLC separations with column dimensions of 250 pm X lo00 mm (%a). With this method, the solvent gradient was formed by several isocratic steps at a very low flow rate. Due to the complex nature of the step gradient elution technique and the inability to precisely reproduce the gradient from run to run, variations in retention time may be anticipated. Nevertheless, a relative standard deviation of less than 2% was reported (25a). An alternative to this is the use of precolumn flow splitting (2.91). Precolumn flow splitting was applied to the 320 pm i.d. packed-capillary system in this work for practical reasons. The split system was determined empirically so that it delivered a measured 3.5 pL/min flow into the sample injector and packed-capillary column. The reproducibility of the solvent gradient as well as the precolumn flow splitting were monitored over a period of 3 days. Figure 2 shows the results from the packed-capillary HPLC separation of the cytochrome C tryptic digest obtained with this system on three consecutive days with UV detection at 214 nm. The solvent gradient was ramped from 5% solvent B to 70% solvent B at a flow setting of 50 pL/min. The actual flow passing through the column was measured to be 3.5 pL/min. Excellent reproducibility of the micro HPLC eluent flow and solvent gradient accuracy was obtained, as evidenced by the close similarity of the three chromatograms observed in Figure 2. This system was used throughout the experiments in this report for the separations using the 320 pm i.d. packed-capillary column. LC/MS with Column Dimensions of 500 pm X 100 mm. T o examine the performance of the ion-spray interface for coupling packed-capillary columns with an internal diameter of 500 pm with APIMS, a synthetic mixture of peptide standards was analyzed. This test mixture consisted of fibrinopeptide A, bradykinin, and substance P in concentrations of 25 pmol/pL for each peptide. Figure 3A shows the packed-capillary HPLC UV chromatogram of this test mixture with 1pL of solution injected on-column. Its corresponding full-scan total ion current (TIC) profile obtained via the
ANALYTICAL CHEMISTRY, VOL. 63,NO. 7, APRIL 1, 1991
735
A
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tryptic digest showing gradient reproducibility over a period of 3 days.
ion-spray interface is shown in Figure 3B. The separation was accomplished by applying a linear solvent gradient from 5% B to 70% B over a period of 60 min. The mass spectrometer was scanned repetitively from 350 to 1400 Da in 7 s. Under such conditions the total run time was less than 25 min. It was not our intention to just "separate" this particular set of peptide samples but to examine the LC/MS performance of the ion-spray interface and the gradient system under packed-capillary HPLC conditions. Comparison of the UV and TIC traces shown in Figure 3A,B reveals preservation of chromatographic integrity by the LC/MS system. No noticeable chromatographic peak distortion was observed. Stable ion currents were obtained throughout the gradient elution. Thus, a wide range of solvent gradient may be applied to "fine tune" the HPLC separation of more complex peptide mixtures such as protein tryptic digests without adversely affecting the stability or performance of the ion-spray LC/MS interface. This is especially important for separating a mixture containing peptides that exhibit a wide range of hydrophobicities. The major advantage of coupling HPLC with MS is the extra dimension of information (e.g., molecular mass) that is obtained by using mass spectrometric detection. Ionization based upon the ion evaporation mechanism is known to be very soft and thus precludes fragmentation. Therefore, molecular weight information is readily accessible from an ion evaporation mass spectrum (15, 18,22,26). From the separation of three peptide standards shown in Figure 3B, we can obtain the mass spectra containing both doubly and triply protonated molecular ions typical for the three peptides (not shown here). Due to the multiple charging feature of this method (15, 22, 27), the molecular weight for each peptide can be determined with a simple equation (27). The experimentally determined molecular weights were 1536, 1060, and 1346 Da for fibrinopeptide A, bradykinin, and substance P, respectively. As a consequence of the soft ionization method
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the mass spectra reveal no structural information. The latter is available, however, if extra internal energy is added to the ions. Application of collision-induced dissociation (CID) or mass spectrometry/mass spectrometry (MS/MS) for obtaining peptide sequence information will be discussed in a later section. It is well documented that reversed-phase HPLC is one of the mlost successful methods for the separation of biomolecules such iw in peptide mapping applications. HPLC with columns of reduced diameter not only enhances the HPLC efficiency and sensitivity but is also desirable for handling small sample sizes (28). Since the ability to form the fine, liquid droplets from electrospray-type interfaces may be enhanced by reducing the liquid flow rate, an overall sensitivity increase from the APIMS viewpoint may be anticipated for LC/MS using packed-capillary columns. Figure 4A shows the full-scan TIC profile from an injection of 50 pmol of human serum albumin (HSA) tryptic digest obtained with this 500-pm X 100-mm
736
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
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doubly charged ions (19,29,30). Thus, the molecular weight determination of peptides from the LC/MS analysis of protein tryptic digests is a straightforward task. When one combines the peptide-mapping capability provided by the high-resolution, reversed-phase HPLC separation with simultaneous molecular weight determination of each separated peptide via ion-spray LC/MS, the technique offers a powerful analytical tool for protein characterization. LC/MS with Column Dimensions of 320 pm X 50 mm. LC separation utilizing columns with an internal diameter less then 500 pm offers advantages which include decreased solvent quantities and hence cost, plus an increase in sensitivity when sample quantities are limited. The very low flow rate required by the packed-capillary HPLC systems is most attractive when MS is considered as the detection method due largely to the fact that no splitting of the LC eluent is necessary in order to achieve the high vacuum condition required by conventional MS (31). Nevertheless, the considerable instrumental problems associated with packed-capillary HPLC systems must be addressed. For a 320-pm x 50-mm packed-capillary HPLC system, the optimum operating flow is in the range 3-5 pL/min. Because peptide mapping by H P L C / W is primarily a comparison-based technique, a high degree of chromatographic reproducibility is required for the separation method employed. The AB1 micro LC pump, which was designed for microbore HPLC operation, was modified to fulfill the lowflow, solvent gradient requirement, and its performance in terms of reproducibility has been examined (see discussion above on gradient formation). Despite the fact that part of the eluent (approximately 46 pL/min) was split to waste, excellent reproducibility of the solvent gradient was achieved and used for all the remaining applications in this report. Figure 5A shows the TIC profile of dephosphorylated bovine dephosphorylated &casein tryptic digest obtained by using the described 320-pm X 50-mm packed-capillary LC/MS system. The separation was accomplished by applying a linear solvent gradient from 10% B to 70% B over a period of 60 min at a measured column flow of 3.5 pL/min. The mass spectrometer was scanned from 350 to 1400 Da in 4.25 s. The injection volume was 1 pL, which represented a total injection amount of 25 pmol. With a flow of 3.5 pL/min, the ion-spray APIMS required no significant changes in the operational parameters, as compared to the MS conditions set for our more typical 40 wL/min flow conditions. The only change made for this experiment was reduction of the nebulizing gas pressure (60 psi reduced to 40 psi) on the ion-spray LC/MS interface. Although the low flow rate applied here was within the operational conditions of pure electrospray, the coaxial nebulizing gas was found necessary for maintaining a stable ion current throughout the gradient packed-capillary HPLC separation. It is well-known that the size of column packing material is one of the factors which determines the retention characteristics of a column. Presently, the most commonly used column packing materials are particles of lo-, 5-, and 3-pm diameter. Generally, the smaller the particle size, the greater the column separation efficiency. In addition, separation can be performed faster without loss of resolution by using short columns packed with small particles. The separation power and peak capacity of these short capillary columns packed with 3-pm particle sizes were adequate for dealing with complex peptide mixtures such as the protein tryptic digest shown in Figure 5A. However, it is important to note that the packing material in this column had a pore diameter of 80 A. The role of pore diameter in HPLC separation of biomolecules is critical. For most organic molecules it is desirable to have a pore diameter of about 100 A. Smaller pore diameters can result in irreversible adsorption. Macromolecules and bio-
ANALYTICAL CHEMISTRY, VOL. 63,NO. 7, APRIL 1, 1991
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polymers such as proteins and large peptides require larger pore diameters generally in the range of 300 A. As a result of the small pore diameter used for this packed-capillary column, some chromatographic peaks were broad but some were sharp. Also observed in the TIC trace (Figure 5A) was the coelution of some peptides under the separation conditions used. No effort was made to optimize the HPLC gradient for this particular separation. The expected increase in overall LC/MS sensitivity was evidenced by the stable total ion current with a high SIN ratio when less sample was injected on-column (compare results from LC/MS with columns of 1-mm (19) and 0.5-mm (Figure 4A) internal diameters). LC/MS results with sample sizes as low as 10 pmol (bovine cytochrome C tryptic digest; data not shown) have been obtained with an excellent S / N ratio. Thus, if a suitable high-quality packed-capillary HPLC column is available, low picomole peptide mapping by packed-capillary LC/MS is feasible and practical. The extracted ion current profile for m / z 742 (Figure 5B) from the LC/MS analysis of dephosphorylated 8-casein tryptic digest demonstrates the additional selectivity offered by MS. Its corresponding ion evaporation mass spectrum is shown in Figure 5C, where the abundant ions at m / z 371 and 742 represent the [M + 2HI2+ and [M H]+ ions, respectively. Its molecular weight was determined experimentally to be 740.5 f 0.7 Da from this LC/MS experiment. Despite the fact that theoretical examination and some applications suggest that high sensitivity, high separation efficiency, and relatively fast analysis times can be achieved by using packed-capillary columns, this technique has been practiced only in a few laboratories worldwide. To overcome
+
its limited acceptance by industry, the technique must demonstrate ease of direct transfer to those separation conditions developed by using conventional 4.6 mm or 1.0 mm internal diameter columns. New method development must be minimized when the column diameter is scaled down to the packed-capillary level. Figure 6A shows the full-scan TIC profile of a crude peptide mixture obtained by microbore LC/MS analysis. The separation was accomplished by applying a linear solvent gradient from 5% B to 70% B over a period of 60 min with the Supelco LC-308 microbore (1.0 mm i.d.) column at a flow of 40 pL/min employing no pre- or postcolumn flow splitting. A predetermined bioactive peptide residue is indicated by the arrow in Figure 6A. The packed-capillary system (320 pm i.d.) used here for the same separation gave the result shown in Figure 6B. The TICS shown in Figure 6A,B clearly demonstrate the ability to directly transfer separation conditions from a 1 mm i.d. microbore column to a 320 pm i.d. packed-capillary column. The small difference in retention time observed for some early eluting peaks was due mainly to the difference in column packing material where the microbore column (Le., 1.0 mm x 100 mm) had 5-pm particles with a 300-A pore diameter and the packed-capillary column (i.e,, 320 pm X 50 mm) had 3-pm particles with 80-8, pore diameter. Figure 6C shows the ion evaporation mass spectrum of the targeted bioactive peptide indicated by the arrow in Figure 6B at a retention time of 15.5 min. The multiple charging feature of the ion evaporation ionization method is clearly evident in this mass spectrum. The experimental molecular weight of this targeted bioactive peptide was calculated to be 3239.96 f 0.01 Da, which correlates well with the data previously obtained by amino acid analysis. Another major concern expressed by some for using packed-capillary columns is the small injection volume allowed by the column. This is true when the separation is carried
738
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
out under isocratic flow conditions. However, the principle of peptide separation by reversed-phase HPLC is an adsorption/desorption process where peptides are adsorbed and remain bound to the stationary phase until a critical percentage of organic modifier in the eluent is reached. Therefore, larger volumes of sample may be injected provided the initial HPLC conditions are set at high aqueous (95% or even 100%) such that the injection "band" may be focused at the beginning of the column. Thus, band broadening due to the large injection volume may be minimized by allowing trace analysis with packed-capillary columns more practical. In fact, sample sizes as large as 5 pL have been injected into a packed-capillary HPLC system without significant increases in band broadening (32). Application of Packed-Capillary LC/MS/MS. Although the molecular weights of separated peptides may be determined by on-line LC/MS, the molecular weight information alone is often not enough for structural identification of analyks. The ability to obtain a peptide's primary structure (Le., its sequence) is vital for positive identification or for characterizing an unknown peptide. MS in tandem configuration (MS/MS) is a powerful tool for this application (32, 33). The advantage of the MS/MS method is that the ability to sequence a peptide is not limited to those peptides with unblocked N-termini, and considerable sequencing time may be saved over the conventional Edman degradation (34). In addition, LC/MS/MS provides on-line structural information for each digest component observed. Since trypsin specifically cleaves peptide bonds a t the carboxyl side of the basic amino acids of arginine and lysine, the nature of these resulting tryptic peptides will be primarily a C-terminus with either arginine or lysine and a free Nterminus (29,30). Hence, ion evaporation daughter ion mass spectra obtained from MS/MS of the doubly charged parent ions of these tryptic peptides provide a special case for peptide sequencing by MS/MS. Since the resulting charges are localized at both ends of the peptide, the additional internal charge repulsion energy provides high collision efficiency for MS/MS (29). The application of this method to peptide sequencing has been demonstrated both by on-line LC/ MS/MS (19, 22, 30) and by off-line direct introduction of protein tryptic digest solutions (29, 30). Nevertheless, there are still many cases where MS/MS analysis of ions other than those in the doubly charged state is required for structural elucidation. An example has been selected in this section to demonstrate the benefit from MS/MS capability with parent ions other than those that are doubly charged. A typical ion evaporation mass spectrum for a separated peptide from a protein tryptic digest was shown previously in Figure 5C, where the doubly and singly charged ions were recorded with high ion abundance. To further examine the structure of this peptide, its singly charged molecular ion at m/z 742 was selected as the parent ion in an MS/MS daughter ion experiment. Figure 7 shows the daughter ion mass spectrum obtained from LC/MS/MS analysis of the sample shown in Figure 5A wherein the mlz 742 parent ion was focused into the collision cell of the triple-quadrupole mass spectrometer. Fragment ions at m / z 70,86, and 120 indicated the possible presence of Pro, Leu/Ile, and Phe, respectively. Ions at m / z 155 and 127, which differ by 28 amu, indicated they were associated B2 and A2 ions. The only combinations of amino acids that would give these A and B ions are Gly-Pro or Pro-Gly. Following this logic, it is reasonable to determine that ions at mlz 625 and 597,512 and 484,399 and 371,302 and 274 were associated B,, and A,, sequence ions. Hence, the tentative sequence of this peptide was read as Gly-Pro-PhePro-Leu/Ile-Leu/Ile-Val or Pro-Gly-Phe-Pro-Leu/Ile-Leu/ Ile-Val. The calculated molecular weight of this peptide from
r"* 742
'7
m/z
Flgure 7. LC/MS/MS mass spectrum from CID of the m l z 742 ion for the C-terminus peptide from dephosphorylated &casein tryptic digest.
the tentatively assigned sequence is in agreement with the molecular weight of the selected parent ion at m/z 742. The amino acid at the N-terminus was still not positively identified. To further elucidate the peptide sequence, the predicted sequence ions of type Y,, in the daughter ion mass spectrum were sought. Daughter ions observed at m / z 231,343,440, and 588 were in good agreement with respect to the predicted Y2, Ys, Y,, and Y5ions, respectively, based upon the sequence tentatively assigned. Nevertheless, the Y6sequence ions which could distinguish the N-terminus sequence of Gly-Pro from Pro-Gly were not observed. Abundant fragment ions observed a t m / z 324, 245, and 211 were assigned as internal acyl ions of Pro-Leu/Ile-Leu/Ile, Pro-Leu/Ile, and Phe-Pro, respectively. They appear to result from nonsequence-specific internal peptide bond cleavage. The loss of a carbonyl group (mass difference of 28 amu) from internal acyl ions of ProLeu/Ile, and Phe-Pro was also observed at m / z 217 and 183, respectively. The fragment ion at m / z 284 was determined to be associated with the loss of the ammonium ion from B3 a t m / z 302. This peptide was found to be the C-terminus peptide of &casein (T-16,amino acid residue 203-209). The ability to deduce peptide molecular weights and sequence information from the peptide components in tryptic digests, or at least gain some structural information, is of considerable importance. CID analysis of a singly or doubly charged tryptic peptide parent ion following on-line HPLC separation demonstrates the potential of this technique for peptide sequencing. CONCLUSIONS High-sensitivity peptide mapping may be achieved by LC/MS using packed-capillary columns. The advantage realized from MS detection is the extra dimension of information available with regard to the molecular mass. Assuming acceptable column technology is available, the only remaining obstacle for performing LC/MS is the formation of a suitable solvent gradient at low eluent flow rates. For packed-capillary columns of 500 pm i.d. the optimum flow is typically around 10 bL/min. This may be satisfactorily accomplished from high-quality commercial micro LC pumping systems. One must also reduce the extracolumn volume associated with the connecting tubing and solvent mixer. No flow presplitting is required in the micro HPLC system described here using the 5 W p m i.d. packed-capillary column. For packed-capillary columns with internal diameters of 320 pm, the optimum flow is usually in the range 3-5 pL/min. In this case, precolumn flow splitting was required in this work for generating a reproducible solvent gradient necessary for peptide-mapping applications. Precolumn flow splitting was accomplished by using a fused-silica capillary split line. The ion-spray LC/MS interface, which we routinely use for microbore LC/MS analyses, may be used for packed-capillary LC/MS without any hard-
ANALYTICAL CHEMISTRY, VOL. 63,NO. 7, APRIL 1, 1991
ware modifications. The only change in operational parameters is the use of a lower nebulizing gas pressure in the ion-spray LC/MS interface. On-line packed-capillary LC/MS for peptide mapping with sample sizes of 10-30 pmol is considered a routine task in our hands. The full-scan detection limit for the described system is believed to be in the low picomole level on the basis of the current data. The MS/MS technique is beneficial for obtaining peptide sequence information. This may be done either by direct infusion of peptide sample or, preferrably, by on-line packed-capillary LC/MS/MS. On-line LC/MS/ MS is particularly well suited for dealing with complex peptide mixtures such as protein tryptic digests. This approach also minimizes the possible ion suppression caused by buffer salts or contaminants which may decrease the overall sensitivity. LC/MS/MS may also avoid the situation in which a selected parent ion may originate from two or more different sample components. A daughter ion mass spectrum from different parent ions with the same m / z value could complicate the interpretation of MS/MS data from unknown analytes. On-line LC/MS/MS could preclude this problem by preseparating the sample components prior to CID. Although direct coupling of packed-capillary columns to a conventional mass spectrometer has been reported, the ease of using an APIMS system has considerable merit. The complete decoupling of the packed-capillary column liquid effluent from the mass spectometer vacuum system in the latter greatly facilitates the LC/MS coupling. Improved sensitivity combined with facile molecular weight determination as well as structural information via LC/MS/MS suggests this combination of analytical technologies offers considerable analytical utility. ACKNOWLEDGMENT We thank Supelco Inc. and LC Packing International for providing packed-capillary columns, Applied Biosystems Inc. for the loan of their micro LC pump, and Sciex Inc. for hardware and research support. LITERATURE CITED Barber, M.; Bordoii, R . S.; Elliott, G. J.; Sedgwick, R. D.; Taylor, A. N. Anal. Chem. 1982, 5 4 , 645A. Sundqvist, 8.; MacFariane, R. D. Mass Spectrom. Rev. 1885, 4 , 421. (a) Denoyer, E.; Van Grieken, R.; Adams, F.; Natusch, D. F. S. Anal. Chem. 1982, 5 4 , 26A. (b) Hercules, D. M.; Day, R. J.; Balasanmugan, K.; Dang, T. A.; Lin, C. P. Anal. Chem. 1982, 54, 280A.
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RECEIVED for review September 10,1990. Accepted December thanks the Eastman Kodak Co. for financial 27,1990. E.C.H. support during his tenure at Cornel1 University.
