Fluorescent peptide mapping with microgram quantities of protein

ANALYTICAL BIOCHEMISTRY 71, 459-470 (1976)

Fluorescent Peptide Mapping with Microgram Quantities of Protein JAMES R. BENSON Durrum Chemical Corporation, 3950 Fabian Way, Palo Alto, California 94303 Received September 24, 1975; accepted November 18, 1975 A microbore column chromatograph utilizing constant pressure eluent and reagent solution pumps is combined with fluorescent detection using o-phthalaldehyde. This yields a versatile, highly sensitive method of separating and detecting peptide fragments obtained from microgram quantities of protein.

Protein chemists must sequence peptide fragments obtained from purified proteins because automatic sequencing of intact proteins is not yet possible. Thus, separation and detection of peptide mixtures is a prerequisite to determining primary structure of the parent protein. Gel electrophoresis, thin-layer chromatography, gel filtration, and ion-exchange chromatography are common means of separating these mixtures. The patterns of separated and partly separated peptides obtained by these techniques form characteristic maps and can often provide valuable information about the original molecule. For example, comparison of tryptic peptide maps of normal and abnormal hemoglobin reveals characteristic structural differences attributable to variation in amino acid composition (1,2), Regardless of the separation method utilized, a major problem lies in the separation and detection of progressively smaller quantities of peptides. Most proteins available in gram quantities have already been sequenced; many proteins of biochemical interest are available only in milligram or microgram amounts. If the structures of these proteins are to be realized, higher sensitivity techniques of analysis must be developed. This report describes a method of attaining high resolution peptide maps from microgram quantities of purified protein. High resolution microbore ion-exchange chromatography techniques modeled after a system described by Hare (3,4) are combined with a recently developed fluorescent assay for primary amines. The fluorescent detection method utilizes ophthalaldehyde (OPA) in the presence of 2-mercaptoethanol; this reagent forms highly fluorescent products upon reaction with primary amines. Use of OPA for this application was first reported by Roth (5) who described reactivity with amino acids but failed to find significant fluorescence with peptides. Benson and Hare subsequently showed that the reagent can be 459 Copyright © 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.

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used successfully to form fluorophors with peptides (6). OPA is preferred over fluorescamine (7) (a similar fluorogenic reagent) because of its stability in water. This property eliminates the need for organic solvents and obviates precipitation problems associated with use of fluorescamine. Another microanalytical method for separating and detecting peptides is that of Herman and Vanaman (8) who modified the peptide monitoring system originally described by Hill and Delaney (9). A modified Technicon autoanalyzer with a conventional reciprocating piston pump was used in an instrument capable of carrying out alkaline hydrolysis of effluent peptides followed by detection with ninhydrin. In comparison with the Herman and Vanaman method, the method described in this report is more sensitive by more than one order of magnitude and is much simpler because neither an alkaline hydrolysis step nor a heated reaction coil is required. METHODS

The Microbore Chromatograph Two basic means of forcing eluents through resin beds exist. (a) The "constant displacement" method typically employs a reciprocating piston pump that repeatedly displaces a constant volume of liquid from a chamber. This system provides a pulsatile, yet constant eluent flow rate regardless of resistance to flow. (b) The "constant pressure" method employs a fixed gaseous pressure applied to the eluent surface in a reservoir; flow rates will change if resistance to flow changes. The chromatograph described below utilizes the constant pressure principle, chosen because of the quiet, pulse-free baselines and improved signal-to-noise ratios that are obtained. A more comprehensive discussion of constant pressure versus constant displacement is found elsewhere (10). A schematic illustration of the chromatograph appears in Fig. 1. All reagents are forced through the system by nitrogen gas pressure controlled by regulators R1 and R2. Teflon tubing (Durrum Chemical Corp.) is used throughout the system and has an inside diameter of 0.8 mm except as noted below. The polystyrene buffer solution reservoirs (Durrum) are inert to all reagents used. A two-way slide valve (Durrum) is connected to each nitrogen inlet and eluent outlet so that any reservoir can be isolated for refilling without depressurizing the entire system. A six-way buffer selection valve (Valco Instruments, Houston, Texas) directs the appropriate buffer solution into the column. A 1-m loop of 0.3 mm i.d. x 0.6 mm o.d. microbore Teflon tubing (Durrum) is connected between the flow meter and the high-pressure sample injection valve (Durrum). The thin walls of this tubing allow some degassing of buffer solutions prior to application of the resin bed. The heated, .stainless steel column contains a 0.20 x 25 cm bed of Durrum DC-4A cation-exchange resin; column effluent plus OPA is

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F L U O R E S C E N T PEPTIDE MAPPING

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FiG. 1. The chromatograph. Schematic illustration of the microbore chromatograph described in the text. Regulators (R] and R2) regulate nitrogen gas pressure provided to the two parts of the system. RI controls nitrogen pressure in the buffer solution reservoirs. Pressure from R2 is carried to the OPA reagent reservoir and to solenoid valves (SOL ] and SOL 2) that control the automatic slide valve and the buffer solution selection valve, respectively. Flowmeter (FM 1) monitors eluent flow rate at sample injection valve (SV) inlet. The jacketed stainless steel column containing an 0.2 × 25 cm bed of Durrum DC-4A cation-exchange resin is maintained at 57°C. Separated peptides in the column effluent react with OPA which is continuously added to the effluent at the three-way manifold (MIX). The reaction takes place instantly at room temperature, and fluorescence is monitored by an Aminco fluorometer. OPA reagent solution passes through filter (F), then through a flow meter (FM2) before entering mixing manifold. A third flow meter (FM3) monitors combined eluent + reagent solution flow rate. Dashed lines represent nitrogen lines; solid lines represent fluid lines.

monitored by an Aminco filter fluorometer (American Inst. Co., Silver Spring, Md.) equipped with a 70/~l flow cell, a Coming 7-60 excitation filter and a Wratten 2A emission filter. (The OPA-primary amine reaction product has an excitation wavelength maximum of 340 nm and an emission wavelength maximum of 455 nm) A 3-m coil of the microbore Teflon tubing at the chromatograph exit creates sufficient back pressure so that degassing cannot occur within the flow cell. A two-pen Honeywell Electronik 19 strip chart recorder, modified by Honeywell to provide a chart speed of 3 in. hr -1, records signals from the fluorometer. The recorder channels are usually set at 100 mV and l0 mV full-scale deflection. Reagent solution passes into a stainless steel column (Durrum) containing resin beads. This creates sufficient flow resistance that pressure regulator R2 can operate in its most precise range. The column also filters particulate matter from reagent solution before it enters the mixing manifold. All connections to valves, manifolds, and tubing throughout the system are made with Durrum "Flarefit" miniature tubing components. In operation, all six buffer-solution reservoirs are pressurized by

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JAMES R. B E N S O N

opening their inlet slide valves and adjusting regulator R1 to an appropriate pressure (typically, 17 to 24 atm). Pressure within the reagent solution reservoir is typically 5 to 7 atm. Approximate eluent flow rates are monitored by flowmeters, but true flow rates are determined by measuring the volume of effluent collected in a graduated cylinder as a function of time. Once a stable baseline is established, sample is injected into the eluent stream. A Durrum eight-function elapsed time programmer actuates the buffer solution selection valve to provide sequential application of buffer solutions. The programmer also shuts off the recorder and terminates reagent flow at the end of the analysis.

Solution Preparation The water used for preparation of all solutions had a specific resistance of at least 16 MI~ cm; it was purified by a commercial deionization system (Aqua Media Co., Sunnyvale, Calif.) that included a prefilter, a charcoal filter, additional organic contaminant sorption cartridge, and two mixedbed ion-exchange cartridges. The water then passed to a flow cell surrounding germicidal ultraviolet lamp, finally passing through a 0.25 micrometer filter. Water conductivity was continuously monitored.

Reagent Solution OPA reagent solution was prepared from Durrum "Fluoropa," a fluorogenic grade ofo-phthalaldehyde. The OPA was dissolved in pH 10.5 borate buffer solution according to instructions provided with the reagent. Several other commercial grades of OPA were tried but were unsuitable because high fluorescent background or erratic baselines resulted with their use. OPA is subject to oxidation; these oxidation products are apparently responsible for the background fluorescence obtained with the other commercial preparations that were tested.

Buffer Solutions Great care was taken in preparation of buffer solutions because of the extremely high sensitivity of the analytical equipment. All glassware was thoroughly cleaned with chromic acid cleaning solution, then rinsed with deionized water. Special care was exercised to avoid touching wetted surfaces because even minute amounts of amino acids transferred from fingerprints could contaminate subsequent analyses (11). The first four solutions each contain 19.6 g sodium citrate dihydrate (Baker) dissolved in 980 ml deionized water to which is added 1.0 ml liquified phenol (Mallinkrodt). The pH values of 4.60, 5.00, 5.45, and 6.25 for the four solutions are adjusted by the addition of reagent grade hydrochloric acid (Baker). The fifth buffer solution contains 1.0 ml phenol and 16.0 g of 50% sodium hydroxide solution (Baker "Analyzed") added to 980 ml water. We add ortho-phosphoric acid (Baker) until pH 7.20 is

F L U O R E S C E N T PEPTIDE MAPPING

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reached (ca. 13 g required). The sixth buffer solution is prepared by adding 4.0 g 50% N a O H , 8.76 g sodium chloride, 1.0 ml phenol and 3.0 g boric acid to 990 ml water and adjusting to pH 9.50 with concentrated HC1. All solutions are 0.20 M in sodium. After preparation, buffer solutions are filtered through a Millipore "Polyvic" 0.5/xm pore filter disc. Because these filter discs leach substances that react with OPA reagent, approximately 2 liters of water should be passed through each new disc before buffer solutions are filtered. The first 100 ml of filtered solution are discarded; the rest is immediately transferred to the appropriate reservoir that is pressurized with prepurifled nitrogen gas.

Sample Preparation When analyses are performed at picomole sensitivity, sample contamination problems can become a major concern. To avoid contamination resulting from sample transfer, all preparations and reactions were carried out in a single vessel. A 1.0-ml borosilicate glass reaction vial (Wheaton Glass Co.) is suitable for this procedure and is just large enough to allow pH monitoring with a Coming microcombination electrode. Prior to addition of protein samples, vials were filled with chromic acid cleaning solution and placed in an oil bath maintained at 120°C to 130°C for about 1 hr. Upon cooling, the vials were thoroughly rinsed with deionized water. Solutions containing 20 to 25/zg of purified protein were added to reaction vials, then total volume was brought to 500/xl with deionized water. Nitrogen gas, bubbled successively through three water-containing bottles, was blown over the solution to displace air in the vial. The vials were then tightly capped and placed in an oil bath (120°C to 130°C) for 20 to 30 min in order to accomplish denaturation. After cooling, the oil was carefully wiped from the outside of the vials. A stock solution containing 1.0 mg T P C K trypsin (Worthington Biochemicals) in 50 ml 10-3 M HC1 was prepared and stored at 4°C and used for subsequent trypsinization. Vials containing denatured protein plus trypsin were always flushed with water-washed nitrogen and capped tightly. After trypsinization, caps were loosened and vials were placed in a lyophilization chamber. Lyophilized samples were dissolved in ca. 30 ttl 10-3 M HC1 and stored at -20°C until analyzed. Samples were aspirated directly from the vial into the 20/A sample loop of the sample injection valve using a 100/.d syringe (Fig. 1).

Analysis Condition Because constant pressure is used to force the eluents through the resin bed, changes in resistance to flow within the system can result in altera-

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tion of eluent flow rate; for this reason, precautions must be taken to reduce or eliminate resistance changes. For example, Millipore filtration of buffer solutions precludes contamination of resin beds by particulate matter. Because cation-exchange resins of the type used shrink or swell depending upon ionic environment, constant molarity buffer solutions are utilized, preventing eluent viscosity changes. Peptides are eluted from the resin bed by increasing p H values of the successive solutions. A carefully monitored eluent flow rate of 6.3 ml hr -1 (linear flow velocity = 3.3 cm min -1) was used in all analyses. Flow rates were checked before each analysis, and eluent reservoir pressure was adjusted as required to maintain a constant flow rate. Analysis conditions are summarized in Table I. The buffer solution program is summarized in Table 2.

Control A "control" sample was prepared by filling a cleaned vial with 500/xl deionized water. The vial was then flushed with water-washed nitrogen, capped, and placed in the hot oil bath with vials containing proteins and was subsequently treated and monitored in exactly the same manner as other vials. The residue remaining after lyophilization was dissolved in 10-3 M HC1. A 20-/zl aliquot was injected onto the resin bed and analyzed. RESULTS

Control Analysis of the control demonstrated that the sample handling technique described above adequately prevented contamination of samples. The resuits are shown in Fig. 2. There are a few small peaks during the first 150 min., but the balance of the chromatogram is free from contaminant peaks. The peak at ca. 140 min is probably ammonia. Baseline shift and erratic behavior beginning at about 600 min is caused by contaminants TABLE 1 ANALYSIS CONDITIONS Resin Resin particle size Resin bed dimensions Column temperature Eluent flow rate OPA reagent flow rate Eluent reservoir pressure (typical) OPA reagent reservoir pressure (typical) Fluorometer (Aminco) range Recorder ranges

Durrum DC-4A cation-exchange resin 9.0 _ 0.5/xm 0.20 x 25 cm 57°C 6.3 ml hr -~ 6.3 ml hr -1 20 atm (300 psig) 7 atm (100 psig) 100× (least sensitive scale) 100 mV and 10 mV full-scale deflection

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F L U O R E S C E N T PEPTIDE M A P P I N G TABLE 2 BUFFER SOLUTION PROGRAM Buffer solution A B C D E F

pH

Anion

Time in System (min)

4.60 5.00 5.45 6.25 7.20 9.50

Citrate Citrate Citrate Citrate Phosphate Borate

180 120 120 60 120 60 660

Total analysis time

contained in the first five solutions that are subsequently eluted from the resin bed by the high pH borate buffer solution. Contaminants present in hydrochloric acid used to adjust buffer solution pH values, and to a lesser extent, in the water used for solution, are responsible (10). Contaminants in the water are nonionic organic substances not removed by the deionization system (unpublished data). These substances either display natural fluorescence or react with OPA to yield fluorophors with excitation and emission relative maxima similar to the OPA + primary amine reaction products. To estimate the sensitivity of the system and to determine the efficacy of the buffer solutions for elution of basic peptides, a standard calibration mixture containing 200 pmol of protein amino acids was analyzed (Fig. 3). Acidic and neutral amino acids are eluted within 30 min with partial resolution of tyrosine and phenylalanine. The major peak at ca. 145 rain is ammonia, followed by lysine, histidine, and arginine at ca. 320 rain. 100

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FIG. 2. Control sample. A glass vial containing 500/zl deionized water was processed with protein samples to determine whether sample handling techniques adequately prevented contamination by OPA reactive substances. The water was "denatured" in a hot oil bath, trypsin was added, and pH was monitored and kept near 9.0 with 10-3 M NaOH. Finally, "lyophilization" was performed and the residue dissolved in 10-3 M HCI. Twenty microliters of this solution were applied to the resin bed and analyzed; resultant chromatogram is shown. A few small contaminant peaks appear during the first 150 min. The large peak at ca. 140 min is probably ammonia.

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FI~. 3. Amino acid standard. A standard calibration sample containing 200 pmol of protein amino acids was analyzed. Only basic amino acids are completely resolved using this protocol. The peak at ca. 145 rain is ammonia, followed by lysine, histidine, and arginine at ca. 320 rain. The small peak appearing before arginine is of unknown composition.

The small peak appearing before arginine was not identified. It was concluded that peptides eluting after arginine (pK~ = 12.5) are extremely basic. Peptides eluting after 500 min probably either contain several basic residues or are very hydrophobic, interacting strongly with the crosslinked polystyrene resin matrix.

SAE- Globin Tryptic Peptides A 20/~1 sample containing tryptic peptides from approximately 10/zg of S-aminoethylated human globin was analyzed (Fig. 4). On the basis of amino acid composition, approximately 30 peptides would be expected from tryptic digestion of the a and/3 chains. This analysis confirms the existence of more than 20 species; however, acidic peptides would not be resolved using this procedure (see Discussion). The sample consisted of peptides obtained from ca. 300 pmol of globin. Assuming no peptide segments are repeated within the protein, each peak then represents 300

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FIG. 4. Tryptic peptides from SAE globin. The chromatogram represents an analysis of tryptic peptides obtained from only 10/xg SAE globin. A 0.20 × 25 cm bed of Durrum DC4A resin was used at a column temperature of 57°C. Eluent flow rate: 6.3 ml hr -1. Recorder sensitivity: 10 mV, full scale deflection. Six buffer solutions were sequentially supplied to the resin bed.

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pmol of unique peptide. The fluorescence exhibited is of the same order as the 200 pmol of amino acids analyzed (Fig. 2), with the exception of the peak at ca. 145 min that is probably ammonia. Thus, amino acids and these peptides appear to produce approximately the same amount of fluorescence when reacted with OPA. Precision

Analytical precision with this method requires a constant eluent flow rate. To test this, analyses of bovine serum albumin (BSA) were accomplished with replicate 20-~1 samples containing tryptic peptides from ca 25/zg of protein (Fig. 5). The resultant peptide maps indicate identical elution patterns; this could only occur if eluent flow rates remained constant throughout both analyses.

DISCUSSION Detection of peptides in column effluents has traditionally involved alkaline hydrolysis followed by reaction with ninhydrin to form the Ruhemann's purple complex that is subsequently photometrically detected. The microbore chromatograph described by Herman and Vanaman (8) employs this detection method. There are advantages to this system. No peptide remains undetected due to a blocked N-terminal amine be-

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FIG. 5. Precision. Tryptic peptides from only 25 /xg of bovine serum albumin (BSA) were analyzed under the same conditions used for other samples. Two replicate BSA samples were analyzed to determine whether the chromatograph provided repeatable results. Identical elution profiles were obtained.

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cause all are converted to free amino acids that react directly with ninhydrin. Sensitivity is enhanced because hydrolysis frees all amino acids to react, whereas ninhydrin would react with only the N-terminus of the unhydrolyzed peptide. Finally, semiquantitative data on the size o f separated peptides can be obtained. A dipeptide, for example, displays less absorbance than an octapeptide because fewer amino acids are available to react with ninhydrin. In spite of these advantages, a number of shortcomings exist. The system is complicated. After addition of sodium hydroxide to the column effluent, the mixture passes into a heated (100°C) reaction coil for approximately 30 min. This procedure hydrolyzes the peptide, but the solution is too basic for successful reaction with ninhydrin, so it must be neutralized usually with acetic or sulfuric acid. After neutralization, ninhydrin is added and the mixture passes into a second heated reaction coil required for formation of Ruhemann's purple. This is finally carried to the photometer where absorbance of the product is monitored at 570 nm. The complexity of the system compromises both sensitivity and resolution. The sample is diluted by addition of all the reagents, and effluent solute mixing is increased by the great lengths of tubing required. Thus, closely eluting peaks, although they may be separated as they leave the .resin bed, may merge before reaching the photometer flow cell. Alkaline hydrolysis can also contribute to magnification of contaminants; for example, a 1-nmol contaminant of a 20-residue peptide appears in the chromatogram as a 20-nmol peak. Thus, absolute purity of samples is required because minute amounts of contaminant peptides can become disproportionately magnified in the final analysis. The fluorescent detection method described in this report is much less complex. OPA reacts instantly at room temperature, so no reaction coils or heated baths are required. Because OPA is soluble and stable in water it can be dissolved directly in the high pH buffer solution needed to optimize fluorescence. Thus only one reagent solution need be added to column effluents and chromatograph plumbing is greatly simplified. It is possible to adapt the chromatograph for preparative separations by installing a stream splitting device at the column outlet to partially divert effluent flow to a fraction collector. For preparative work with large columns, advantage can be taken of the high sensitivity of the analytical system so that only a very small fraction of the column effluent need be removed for analysis. As long as the OPA reagent solution is of sufficient buffering capacity, any number of eluent formulations can be utilized. The protocol described in this report will achieve separation of neutral and basic peptides from enzymic digests of purified proteins. However, acidic peptides (those eluting during the first 30 min in the chromatograms shown) can be similarly separated using anion-exchange resins and a sequence of buffer solutions

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of decreasing pH values. Although sodium buffer salts were used in these experiments, volatile buffer solutions would work as well. It should be noted that sequencing of collected purified peptides demands desalting of individual fractions if the former eluent formulations are utilized. Satisfactory peak heights were obtained by Herman and Vanaman with 5 nmol of protein digest, but only after effluent peptides had been hydrolyzed by sodium hydroxide. In contrast, using OPA detection, satisfactory peak heights were obtained without hydrolysis of peptides starting with only 300 pmol of digested protein. Much greater sensitivity is possible. The Aminco fluorometer used in these experiments was operated at the least sensitive setting. A 1000-fold signal amplification is theoretically possible with that instrument. Because constant pressure pumping insures quiet, pulse-free baselines, sensitivity of detection is only limited by buffer solution contaminants that naturally fluoresce or are capable of reacting with OPA. These contaminants, if present in the first buffer solutions deposited on the resin bed, are bound to the resin only to be later eluted by higher pH solutions subsequently supplied to the bed. Their elution creates baseline artifacts that interfere with sample peaks. Although picomole quantities of primary amines are readily detected using solutions prepared from commercially available reagents, dramatic improvement in sensitivity could be achieved with improvements in reagent quality. Investigations into the preparation of such purified reagents are underway in this laboratory. The major disadvantage to OPA detection is the inability of the reagent to form fluorophors with secondary amines. Consequently, peptides containing N-terminal proline or hydroxyproline, or with a blocked Nterminus cannot be detected. This is usually not a problem with tryptic digests because trypsin does not cleave lysine or arginine residues that are adjacent to proline (12). However, with other enzymic digests or with chemical cleavages of proteins, N-terminal proline peptides could be formed. If detection of every peptide is essential, an alternate analytical technique must be employed. Benson and Hare have shown that secondary amino acids can be oxidized to primary amines with hypochlorite, allowing subsequent reaction with OPA (manuscript in preparation). Whether this method can be used with peptides has yet to be shown, but the technique seems likely to succeed. ACKNOWLEDGMENTS I thank Dr. P. E. Hare for initially supplying much of the apparatus for the constant pressure system he developed and for providing many needed consultations concerning its operation. I also thank Dr. Ralph Bradshaw, in whose laboratory I prepared the purified human globin and who happily consulted with me during the early stages of this work. Finally, ! thank Dr. Irving Weissman who graciously supplied the purified bovine serum albumin.

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REFERENCES 1. Jones, R. T. (1964) Cold Spring Harbor Syrup. Quant. Biol. 29, 297. 2. Clegg, J. B., Naughton, M., Weatherall, D. (1966) J. Mol. Biol. 19, 91. 3. Hare, P. E. (1969) in Organic Geochemistry, Methods and Results (Eglinton, G., and Murphy, M. T., eds.), p. 438, Springer-Verlag, New York. 4. Hare, P. E. (1972) Space Life Science 3, 354. 5. Roth, M. (1971)Anal. Chem. 43, 880. 6. Benson, J. R., and Hare, P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619. 7. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., Weigele, M. (1972) Science 178, 871. 8. Herman, A. C., and Vanaman, T. C. (1975) Anal. Biochem. 64, 550. 9. Hill, R. L., and Delaney, R. (1967) in Methods in Enzymology (Hirs, C. H. W., Colowick, S. P., and Kaplan, N. O., eds.) Vol. 11, p. 339, Academic Press, New York. 10. Benson, J. R. (1975) in Instrumentation in Protein Sequencing (Perham, R., ed.) Academic Press, London. 11. Hamilton, P. (1965) Nature 205, 284. 12. Schroeder, W. A. (1968) The Primary Structure of Proteins, Harper & Row, New York.