ANALYTICALBIOCHEMISTRY

188,72-81(1990)

Optimization of Immobilized Enzyme Hydrolysis Combined with High-Performance Liquid Chromatography/Thermospray Mass Spectrometry for the Determination of Neuropeptides Robert

D. Voyksner,*T1

David

C. Chen,t,2

and Harold

E. Swaisgoodt

*Analytical and Chemical Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina and TDepartment of Food Science, North Carolina State University, P.O. Box 7624, Raleigh, North Carolina 27695

Received

October

27709,

13,1989

Peptidases, including chymotrypsin, thermolysin, trypsin, Vs protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with HPLC/ thermospray MS for the analysis of neuropeptides. The optimal operating conditions for each immobilized enzyme bioreactor were determined. Optimal hydrolysis usually occurred at the highest percentage of aqueous solution in the mobile phase at pH 7-8 and 40-50%. Often post-HPLC column addition of aqueous solutions before the bioreactor could improve activity and thermospray sensitivity without changing the HPLC separation. Enzymatic hydrolysis requirements were compatible under conditions for HPLC separation and thermospray MS detection of the selected neuropeptides. Synthetic a-, /3-, and y-endorphins were the primary neuropeptides used to evaluate on-line immobilized enzyme bioreactor/MS. HPLC followed by peptidase hydrolysis produced characteristic hydrolysis products for confirming the peptides’ identity using thermospray MS detection. Furthermore, the peptide formed from enzymatic hydrolysis resulted in a MS ion current lo-40 times higher than that of the [M + 2H12+ ion for unhydrolyzed &endorphin. The increased sensitivity achieved for detecting the hydrolysis products permits detection and quantitation of synthetic peptides down to 800 fmol. o 1990 Academic PWS, I~C.

The use of immobilized forms of enzymes offers a number of advantages over their nonimmobilized forms. 1 To whom correspondence should be addressed. 2 Current address: Department of Molecular Genetics and Microbiology, Burroughs Wellcome Co., 3030 Cornwallis Rd., Research Triangle Park, NC 27709.

These advantages include (a) adaption for automation and continuous flow, (b) potentially higher enzyme stability than soluble forms, (c) controlled reaction kinetics, and (d) fewer reaction by-products (1). Use of peptidases as immobilized forms is particularly advantageous with respect to the above points because autolysis does not occur; therefore, the immobilized form is significantly stabilized and the reaction products are not contaminated with enzyme or autolysis products (2,3). Immobilized peptidases have been used to probe the sequence of peptides and proteins. The combination of immobilized peptidase with MS could aid in specific and sensitive detection of peptides and proteins. Techniques including FAB (4,5), tandem MS (MS/MS) (6,7), and thermospray MS (8-11) have been used to detect peptides in a variety of matrices. Typically these MS techniques are limited in the sequence information that can be provided at picomolar levels for most peptides above molecular weights 2000 to 3000 Da. The limitations in specificity (amino acid sequence information) and sensitivity can be partially overcome by the combination of immobilized enzyme bioreactors with HPLC/MS. Although the possibility of using immobilized peptidases in-line with thermospray MS has been demonstrated (12-15), its capabilities can be further developed. For example, maximum stability and specific activity of the immobilized forms have not been developed, particularly stability in the presence of various organic solvents, various pHs, and buffers used for HPLC resolution. Endopeptidases in addition to trypsin and chymotrypsin can be immobilized to aid in peptide identification. Also, techniques for precise control of the extent of reaction and for incorporation of numerous other peptidases that should prove useful for structural analysis have not been optimized.

72 All

Copyright 0 1990 rights of reproduction

0003-2697/90 $3.00 by Academic Press, Inc. in any form reserved.

DETECTION

OF

ENDORPHINS

USING

This paper reports the employment of several different immobilized endopeptidases (chymotrypsin, thermolysin, trypsin, and Vs protease) and exopeptidases (carboxypeptidases A, B, and Y), in combination with HPLC/thermospray MS for the identification of synthetic endorphins. Factors affecting enzyme activity were characterized to determine compatibility with HPLC separation and thermospray MS detection. Synthetic endorphins were chosen for this study, with ,&endorphin receiving the most attention, because they have been found in the central nervous system (16,17). Endorphins are contained in the carbaryl-terminus of proopiomelanocortin, with P-endorphin containing 31 residues (mw 3463), y-endorphin containing 17 residues (mw 1859), and Lu-endorphin containing 16 residues (mw 1745). Previous work demonstrated HPLC/thermospray MS techniques for the detection and quantitation of these endorphins (15). This paper reports the use of immobilized enzyme bioreactors after a HPLC separation, a configuration previously not investigated, for thermospray MS detection and validation of target endorphins in plasma and cerebrospinal fluid. MATERIALS

AND

IMMOBILIZED

ENZYME

BIOREACTORS

73

the TNBS test indicated complete succinylation of the amino groups on the bead surface. Immobilization of enzymes. Enzymes (carboxypeptidases A, B, and Y, chymotrypsin, thermolysin, trypsin, and Vs protease) obtained from Sigma Chemical Co. were used for immobilization. About 20 mg (6-8 X lop7 mol) of each enzyme was dissolved in 0.1 N phosphate buffer, pH 7.0, and placed in a 10 X 75mm test tube with 1 g of succinylamidopropyl glass beads. After degassing, 0.02 pmol of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma Chemical) was added to the tube, which then was sealed with paraffin and rotated at 4°C overnight for simultaneous activation/immobilization (18). The immobilized enzyme was removed from the tube and rinsed with deionized water (1 liter), followed sequentially by 2 M urea (500 ml), 2 M NaCl (500 ml), and Tris buffer, pH 7.5, containing 0.2% sodium azide and 20 mM CaCl, (200 ml) (Sigma Chemical Co. and Aldrich Chemical). The beads were stored at 4°C (21). About 0.18-0.2 g of immobilized enzyme beads was used to slurry pack stainless-steel (10 cm X 2.1 mm) column reactors.

METHODS

Enzyme Immobilization

Assay of Immobilized

Bead preparation. The controlled-pore glass beads (mean pore size 0.2 pm), obtained from Electronucleonits Corp., were cleaned by heating with concentrated nitric acid over a boiling water bath for 1 h (18). The beads were then rinsed exhaustively with deionized water and the fines decanted several times. The beads were dried in a 100°C oven. Aqueous silanization. A 10% aqueous solution of aminopropyltriethoxysilane (Sigma Chemical Co., St. Louis, MO) was prepared and adjusted to pH 4 with 6 N HCl. This solution was degassed, added to the dried beads, and incubated for 3 h at 70°C in a water bath (18). The excess solution was decanted and the beads were placed overnight in a vacuum oven at 100°C. The aminopropyl beads were then washed exhaustively with deionized water and dried in a 70°C oven overnight. A trinitrobenzenesulfonate (TNBS)3 (Sigma Chemical Co.) (l&19) test was used to verify (appearance of an orange color) the presence of amino groups on the glass surface. Nonaqueous succinylation. A solution of 10% SUCcinic anhydride (Sigma Chemical Co.) and 1% triethylamine in acetone was added to the aminopropyl-silanized beads (20). This mixture was allowed to react for 1 h at room temperature, with nitrogen bubbling for mixing. The succinaminopropyl glass beads were then washed with acetone and dried at 70°C. A colorless solution from

Activity of each immobilized preparation was determined using a microrecirculation reactor that allowed initial rates to be determined on small quantities of beads with negligible external diffusion limitation (22). The activities of the bioreactors, determined using standard literature methods, were as follows: carboxypeptidase A, 15 pmol/min/g of beads (23,24); carboxypeptidase B, 25 pmol/min/g of beads (23,25); carboxypeptidase Y, 2 pmol/min/g of beads (26); chymotrypsin, 35 pmol/min/g of beads (21,23,27); thermolysin, 9 pmol/ min/g of beads (28,29); trypsin, 235 pmol/min/g of beads (23,30); and Vs protease, 23 ymol/min/g of beads (31).

3 Abbreviations brospinal fluid;

used: TNBS, trinitrobenzenesulfonate; RIA, radioimmunoassay.

CSF,

cere-

Endorphin

Enzyme Activity

Samples

Synthetic endorphin standards (a-, @-, y-, and leu5-/3endorphin) (Sigma Chemical Co,) were prepared by dissolving 0.25 mg of each in 250 ~1 of water:methanol:trifluoroacetic acid (80:20:1). Each standard was diluted with 0.1 M ammonium acetate to make standard solutions from 0.1 to 300 pmol. The standards were stored in the freezer when not in use. Solvents used for dissolution extraction and HPLC separation (isopropanol, methanol, and acetonitrile) were supplied by Fisher Scientific. Water was obtained in-house from a distillation and resin purification system (Millipore). Canine cerebrospinal spinal fluid (CSF) samples (1 ml) spiked with synthetic endorphins (3-170 pmol) and an internal standard (leu5-/3-endorphin) (70 pmol) were extracted using a Sep Pak (Waters Associates, Milford,

74

VOYKSNER,

CHEN,

MA) cartridge procedure previously described (11,32). The samples were reconstituted in 0.1 M ammonium acetate before HPLC/immobilized enzyme bioreactor/MS analysis. This sample preparation procedure resulted in 70-80% recovery of spiked synthetic endorphins (11). HPLCIMS The HPLC instrumentation consisted of one Model 590 pump and Model U6K injector (Waters Associates, Milford, MA). The endorphins were separated on a BioRad (Richmond, CA) Hi-Pore RP-304 or Hi-Pore RP318 5-Km particle diameter reversed-phase column (25 cm X 4.6 mm). The isocratic solvent system used for the separation was either 25 or 27.5% isopropanol in aqueous ammonium acetate (0.1 M), adjusted to a pH between 7 and 8 with ammonium hydroxide, at a flow rate of 1.0 ml/min. The exact percentage of isopropanol used depended on the endorphins present in the sample and on matrix components that could interfere with the analysis. The separation of mixtures of endorphins (i.e., (Y-, p-, P-fragment l-27, and y-endorphin) was accomplished using 25% isopropanol. The quantitation of P-endorphin using leu5-@-endorphin internal standard was accomplished using 27.5% isopropanol. Retention times of pendorphin using these conditions ranged from 17 min (27.5% isopropanol) to 35 min (25% isopropanol). The HPLC/MS was performed on a Finnigan MAT 4800 quadrupole mass spectrometer with a Finnigan MAT thermospray interface (San Jose, CA). The interface was operated with a source temperature of 27O”C, a vaporizer temperature of 90-lOO”C, and a repeller setting between 60 and 80 V. Positive ion detection without use of the filament or discharge proved most sensitive for detection of the endorphins. The mass spectrometer was programmed to scan the range m/z 250-1800 in 1.5 s. Multiple ion detection was employed for quantitative analysis of the hydrolysis fragments of the endorphin and the internal standard (leu5-P-endorphin) in CSF. The [M + H]+ ion of the N-terminus fragment for the sample and internal standard (m/z 1001) was monitored for 0.5 s each. The mass calibration was verified with polypropylene glycol (a mixture of average molecular weights 1000,2000, and 4000). RESULTS

Optimization

AND

DISCUSSION

of Conditions

for Enzymatic

Hydrolysis

The effective use of immobilized enzyme bioreactors combined with HPLC/MS must consider factors that influence the quality of data generated. The immobilized enzyme bioreactors, HPLC, and MS all have optimal conditions that overlap, permitting sensitive and specific detection of sample peptides. Previous work has demonstrated that thermospray MS sensitivity was optimal in solutions containing high percentages of aque-

AND

SWAISGOOD TABLE

1

Enzyme Specificity for Hydrolysis Enzyme Endopeptidase Trypsin Chymotrypsin V8 protease Thermolysin Exopeptidase Carboxypeptidase A Carboxypeptidase B Carboxypeptidase Y a Secondary b Preferential

of Peptide Bonds

Peptide bond(s) LYS, Ax

Phe, Tyr, Trp (Leu, Met, Asn)” Glu, Asp Leu, Ile, Phe, Met, Ala, Val (Tyr, Ser)” C-Terminal branched C-Terminal primarily C-Terminal amidated

Gly, Thr,

hydrolysis (aromatic or side chain amino acids) b hydrolysis (basic amino acids Lys and Arg) b hydrolysis (Pro, and other amino acids) b

amino acids. amino acids,

ous ammonium acetate within a narrow vaporizer temperature range (33-34). To maintain optimal thermospray sensitivity, HPLC conditions using high percentages of water in isopropanol could be used to separate the endorphins (33). An isocratic separation was chosen to allow more precise optimization of the thermospray vaporizer temperature and to improve thermospray stability. On-line immobilized enzyme bioreactor conditions were evaluated to determine factors influencing the selectivity and enzyme activity over a range of conditions used in HPLC/MS analysis of peptides. The main parameters of concern for on-line immobilized enzyme bioreactor operation are specificity and activity. Specificity is determined by the characteristics of the enzyme and MS analysis. Table 1 lists the amino acid residues for the peptide bonds acted upon by each enzyme in this study. A priori knowledge of the polypeptide’s primary structure helps determine the choice of an appropriate enzyme for hydrolysis. The thermospray spectrum for unhydrolyzed /3-endorphin is given in Fig. 1A. The spectrum consists of only [M + 2H12+, [M + H + Na12+, and weak [M + 3H13+ ions. The mass range l-imitation of the quadrupole allows only the detection of ions to m/z 1800; therefore, the molecular ion (m/z 3464) could not be detected. The spectra shown in Fig. lB-1E illustrate the different patterns obtained for @endorphin after one pass through an immobilized enzyme bioreactor. Hydrolysis by chymotrypsin or thermolysin resulted in a complex, information-rich spectra due to the greater range of peptide bonds hydrolyzed by these two endopeptidases. The chymotrypsin column apparently exhibited a high activity because hydrolysis of peptide bonds of lower specific activity for chymotrypsin (Leu, Met, Asn) were detected. Thermospray spectra for ther-

DETECTION

OF

ENDORPHINS

USING

molysin hydrolysis proved quite complex due to the incomplete hydrolysis of all peptide bonds for which it is selective. Trypsin and Vs protease hydrolysis spectra were more straightforward to interpret because these enzymes hydrolyze fewer peptide bonds and complete hydrolysis was observed. In each case, enzymatic hydrolysis produced structurally confirming ions absent from the unhydrolyzed thermospray mass spectrum. The thermospray spectra of the hydrolysis products from ,& endorphin exhibited [M + H]+ and sometimes [M + Na]” ions (M is the molecular weights of the enzymolysis peptide). The higher molecular weight hydrolysis products (~500 molecular weight) exhibited more intense [M + Na]+ signals than those at lower molecular weights. The activity of the immobilized enzyme bioreactor plays an important role in the quality of the spectra. Immobilized enzyme activity is dependent on pH, temperature, solvents, and buffers used for the hydrolysis. Extremes of any one condition can irreversibly destroy bioreactor activity. Hydrolysis at conditions far from optimal can lead to no hydrolysis or partial hydrolysis of a peptide, limiting the useful information for the peptide. In order to successfully employ an immobilized enzyme bioreactor on-line with HPLC/thermospray MS, the range of conditions that result in high bioreactor activity and are suitable for HPLC separation and thermospray MS operation must be defined. The effect of pH on the activity of the immobilized enzymes studied is shown in Fig. 2. Results indicate that the endopeptidases have an optimum pH between 7.5 and 8.0, carboxypeptidase A has an optimum pH from 7.0 to 7.5, and carboxypeptidase Y has an optimum pH from 6.0 to 6.5. In the caseswhere multiple enzymes are employed, solution pHs in the range 7-7.5 appeared optimal and did result in less than 50% loss in activity for an enzyme such as carboxypeptidase Y. When the immobilized enzymes were exposed to a pH + 1 to 2 units from the optimal range for a period up to 4 h, 90% of the enzyme activity could be regenerated when conditions were returned to optimum, demonstrating the stability of the immobilized enzyme. However, exposure to pH above 10 and below 4 often resulted in an irreversible loss in enzyme activity. These experiments indicated that typical HPLC separation conditions for peptides using 0.1% trifluoroacetic acid (pH < 2) were not permissible. The use of isopropanol in aqueous ammonium acetate adjusted to pH 7-7.5 with ammonium hydroxide was suitable for HPLC separation and thermospray operation, and was optimal for enzymatic hydrolysis. The temperature of the immobilized enzyme bioreactors affects the enzyme activity according to the activation energy of the reaction, the effects on mass transfer, and the effects on enzyme structure. Column temperatures at 37-50°C could increase activity by a factor of 2

IMMOBILIZED

ENZYME

BIOREACTORS

75

to 3 over room temperature. There was no observed loss in enzymatic activity for extended times at 37 or 50°C temperatures. Enzymatic activity is strongly dependent on the choice and percentage of organic solvent in an aqueous solution. To achieve suitable resolution and elution, organic modifiers must be added to an aqueous solution for reversed-phase chromatography. Isopropanol, acetonitrile, and tetrahydrofuran (25% (v/v) in HzO) solutions do not appreciably affect the activities of the bioreactors. Use of ethanol or methanol yielded nearly a factor of 2 loss in activity for some enzymes including trypsin and carboxypeptidase B. Acetone exhibited the greatest denaturing power resulting in nearly complete loss of activity. Previous work has shown isopropanol to be suited to the chromatography of endorphins (11); therefore, this solvent was the first choice for further evaluation. The extent that isopropanol reduces enzyme activity when added to an aqueous solution at various concentrations is shown in Fig. 3. The isopropanol range O-30% appears to be suitable for bioreactor operation (Fig. 3). In the isopropanol range O-15%, similar or higher enzyme activity was usually observed, perhaps due to increased structural flexibility of the enzyme when low percentages of isopropanol are present. Thermolysin was the exception, showing drastic a decrease in activity with low percentages of isopropanol. Solutions containing 15-30% isopropanol resulted in lossesof enzyme activity (typically less than 50% loss of activity). At isopropanol percentages greater than 30%, the enzyme activity further decreased presumably due to structural changes in the enzyme. Isopropanol proportions of greater than 50% were not explored due to low enzyme activity and thermospray MS sensitivity (33). However, the activity of the immobilized enzyme exposed to high percentages of isopropanol appeared recoverable. Nearly complete activity was recovered following exposure to 50% isopropanol in aqueous ammonium acetate solution for periods up to 24 h. Hence, structural changes induced by concentrations less than 50% are apparently reversible. The best enzymatic activity is usually found in aqueous solvents containing Tris or phosphate buffers. However, volatile buffers must be substituted for these buffers in thermospray MS operation. The effect of various volatile ammonium ion buffers on enzyme activity is presented in Fig. 4. Most of the buffers show minimal loss of enzyme activity relative to the Tris buffer. Ammonium carbonate or bicarbonate appears to be the poorest choice for carboxypeptidase A, with a loss of more than half the enzyme activity relative to Tris buffer. The use of ammonium carbonate and bicarbonate also resulted in significant losses in activity for carboxypeptidase B. Enzyme activity in ammonium acetate, formate, and hydroxide was similar (+20%) to that in Tris buffer. On the basis of these results and earlier work evaluating

76

VOYKSNER,

CHEN,

AND

SWAISGOOD

4

*

TL, m$442

TL, mw 1007

4

TL, mw 1154 .

C

A

‘;;_:, 900

loo

TL, mw 1320 TL,mw 1120

*

[TL, + H]+[T$

700

loot 50

1000

1050

YGGF 4

c,442

1 M 1 TSEKSGTP -

C*mwl560 < ^ _A^..

~

L 1I VTLF

1I KNAIIKNAYI

IC, + HI+

[Cs + HI+ 461

443

[Tb+Hl+ I 1321 I 01 1300 1350

1750

YGGFMTSEK

w-+--w--,

+ +

D

1

T2 mw 1132

T1

IT,, + HI+

K, + HI+

[C, + HI + 476

NAIIK

NAYK

T3 mw 557

T4 mw 494

KGE

IT, + HI+

I 1450

1500

900

1150

1550

1200

mh

FIG. 1. Thermospray mass cm X 2.1 mm) bioreactor, (C) X 2.1 mm) bioreactor, and (E) of P-endorphin in aqueous 0.1

spectra of P-endorphin after one pass through after one pass through M ammonium acetate

950

1000

1050

1100

50

lC, + HI+ 1561 I 1400

I 1550

1500

~-~

50

1350

1460

574

600 650 100 r fT2 + HI+

0

m/z

SQTPLVTLFK

mw 1016

100

1150 I

I 1400

KKGE

‘C,i!$i&O

*

mw 573 -C,mw969-Csmw476

100 rC,

50

1700

.

Ir3mw IIY”

1+ I~~ B

1650

1100

[TL, +Hl’ 1434

5o

1600

+H]+

~‘~~~+H

950

1550

w

* -TL,mw476 TL, mw 591

I (fTL, +H]+ 1 [TL8+HJ+

‘fj/ri; 1500

mw 69a

TL,, mw 1049

TB

TL, mw 1433

4 M

?L,,

4

*

1250

1300

1350

1400

1460

ml2

(A) analyzed a thermolysin a Vs protease under identical

directly without hydrolysis, (B) after one pass through a chymotrypsin (10 (10 cm X 2.1 mm) bioreactor, (D) after one pass through a trypsin (10 cm (10 cm X 2.1 mm) bioreactor. All spectra were acquired for 1 nmol injection conditions.

thermospray sensitivity versus buffer (33), ammonium acetate was chosen for on-line enzyme-catalyzed hydrolysis. Additional experiments indicated that ammonium acetate concentrations between 0.1 and 1 M vield the highest immobilized enzyme activity. Buffer concentrations at 0.01 M or lower resulted in a factor of 2 or more loss in enzyme activity as well as reduced thermospray MS sensitivity. Buffer concentrations greater than 0.1 M show drastic reduction (factor of 10) in activity for V8 protease and carboxypeptidase A, whereas the activity

for other enzymes such as trypsin mained about the same. Effects of Bioreactor

Activity

and chymotrypsin

on Extent

re-

of Hydrolysis

Activity of the immobilized enzyme bioreactor is dependent on the operational conditions, as previously shown, as well as characteristics of the immobilization process, support, column size, and sample concentration. Because the objective of the enzyme-catalyzed hydrolysis is to generate characteristic peptide fragments,

DETECTION

YGGFMTSE

,oo,

1150

1200

USING

*

mw 2589

mw890

0’

ENDORPHINS

KSQTPLVTLFKNAIIKNAYKKGE * VZ

-T-+

E

OF

1250

1300

,

1350

1400

1450

m/z

FIG.

l-Continued

the loss or gain in column reactor activity must be correlated with the information generated from thermospray MS. Comparison of thermospray mass spectra obtained following tryptic hydrolysis of P-endorphin under optimal conditions (pH 7.5,37”C, 100% aqueous ammonium acetate) (Fig. 1D) to those obtained under conditions necessary for HPLC separation (pH 7.5, 37°C 27.5% isopropanol in aqueous ammonium acetate) (Fig. 5) illustrates the effect of lowering enzymatic activity. In this example, the incomplete hydrolysis is beneficial because tryptic fragments T, and T, are detected along with the molecular ion of cy-endorphin. The observed decrease in the extent of hydrolysis can be compensated for by adding aqueous ammonium acetate into the HPLC mobile phase (post-HPLC column) and by recycling a peptide through the bioreactor. In the

160

-. . . . em.-.. --...----------

Trypsin Chynmrypsin Carboxypeptidare Carbaxypeptidare “-8 Protease Thermolydn

-

140 120

IMMOBILIZED

ENZYME

77

BIOREACTORS

configuration where the immobilized bioreactor is after the HPLC column, post-HPLC addition of aqueous ammonium acetate can improve bioreactor activity, and improve thermospray sensitivity without changing the HPLC separation (35). For example, a HPLC separation for endorphins (0.4 ml/min) employing 25% isopropanol can be adjusted to 6.25% isopropanol through postcolumn addition of aqueous ammonium at a flow rate of 1.2 ml/min. Postcolumn aqueous buffer addition plays a major role in achieving optimum activities for peptidases, like thermolysin, which are drastically influenced by solvent composition. Furthermore, the extent of hydrolysis can be improved through a closed-loop recycling of a volume collected at a particular retention time, for a period of time using a peristaltic pump. After a period of recycling, the entire volume is introduced into the thermospray MS system for analysis. The increase in the extent of ol-endorphin hydrolysis achieved by adding post-HPLC column aqueous buffer and by recycling for 8 min (estimated 150 cycles through the immobilized column) through a carboxypeptidase A, B, and Y (1:l:l) bioreactor is shown in Fig. 6. These procedures increased the extent of hydrolysis to allow the determination of the sequence of six more amino acids than could be determined without postcolumn aqueous buffer addition or recycling. Thermospray

MS Sensitivity

A major advantage of combined HPLC/immobilized enzyme bioreactor/thermospray MS is improved sensitivity. Hydrolysis of an endorphin with an endopeptidase can drastically reduce the molecular weight of the peptide. The lower molecular weight hydrolysis products appear to be subject to higher ionization, transmission, and detection efficiency in quadrupole mass spectrometers compared to the nonhydrolyzed peptide. Clearly, a gain of a factor 40-50 in sensitivity could be achieved by

+--*-*---.-.a ----- - - --.... .. .. . . ..

160 A B

140 120 3 .c + E

Chymotryprm CarboxyPeptidase Carboxypsptidare V-8 Proteare Thermolysin carbaxypePtidare

A B y

100 80 60

0 5

6

7

8

9

PH

FIG. 2. Immobilized enzyme activity at various solution pH values. All activities are normalized to 100%. pH was adjusted by using ammonium hydroxide or acetic acid. Activities were assayed in the microrecirculation reactor using the standard synthetic substrates.

0%

12.5%

25%

37.5%

50%

lropropanol (%) FIG. 3. Bioreactor activity after exposure to various concentrations of isopropanol in aqueous ammonium acetate (0.1 M). Activities are normalized to 0% isopropanol. Assays were performed in the microrecirculation reactor using the standard synthetic substrates.

VOYKSNER,

78

m

CHEN,

Carboxypeptidase

B

100

80 .g B .g

80

2 40

AND

SWAISGOOD

tide identification or verification. There are a number of ways of combining the immobilized enzyme bioreactor, HPLC, and MS detection for peptide analysis. Use of an endopeptidase bioreactor prior to HPLC separation and MS detection will enable separation of each hydrolysis fragment for identification (13,15). Typically, this bioreactor configuration can only be used on purified samples because no separation or column cleanup is performed before hydrolysis. The information gathered from this configuration can help identify or confirm the identity of a peptide. Furthermore, the addition of an exopeptidase bioreactor after the HPLC column will permit sequenc-

20

ai Trio

Ammonium carbonme

Ammonium Bi~WZlllilte Buffers

Ammonium Acmme

Ammonium Hydroxlde

Ammonium FOllIlaiS

(0.1 M, pH 7.5)

FIG. 4. Trypsin, Vs protease, carboxypeptidase A, and carboxypeptidase B activities measured in aqueous solution containing 0.1 M of each buffer. The enzymatic activities are normalized to the Tris buffer value.

monitoring trypsin fragment T3 or T, (mw 557 and 494, respectively) of P-endorphin versus the detection of the [M + 2H]‘+ ion of P-endorphin. A gain of a factor 10 in sensitivity can be achieved by monitoring trypsin fragment T1 or Tz (mw 1018 and 1132, respectively) of pendorphin versus the detection of the [M + 2H]‘+ ion of @-endorphin.

Application of HPLC/Immobilized BioreactorjThermospray MS

i ‘;!oo too,

,150

1200

,260

ISW

1350

” ‘OOE

Enzyme h

The combination of immobilized enzyme bioreactor with HPLC/thermospray MS can be very useful in pep-

1060,100

1

‘:I_

100

460

500

650

Km

660

700

750

r

YGGFMTSEKISQTPLVT -100

r

ITT2+ HI+

Tl

TZ

15

mh

FIG. 5. Trypsin bioreactor/HPLC/thermospray MS spectrum of cyendorphin obtained using a 10 cm X 2.1 mm trypsin bioreactor with 25% isopropanol in aqueous ammonium acetate solution. The spectrum shows incomplete hydrolysis due to loss of bioreactor activity in the presence of isopropanol.

FIG. 6. Enzymatic hydrolysis of cY-endorphin using a carboxypeptidase A, B, and Y (1:l:l) bioreactor showing sequential C-terminus amino acid cleavages. (A) Spectra of ol-endorphin following one pass through the immobilized enzyme bioreactor (less than 10 s of exposure) in a 25% isopropanol aqueous ammonium acetate solution. (B) Spectra for cY-endorphin following recycling through the bioreactor for 8 min (about 150 cycles through column). The percentage of isopropano1 was reduced from 25 to 6.25% through post-HPLC column buffer addition (1.2 ml/min).

DETECTION

OF

ENDORPHINS

USING

ing of the separated endopeptidase products, as previously reported by Stachowiak et al. (14) and Kim et al. (15). Reversal of the immobilized enzyme bioreactor and HPLC column (HPLC/immobilized enzyme bioreactor/ thermospray MS), which has not been previously explored, can prove useful in gaining specificity for detection of individual peptides in a complex matrix. HPLC separation followed by immobilized endopeptidase bioreactor and thermospray MS detection was the most logical configuration for the analysis of peptides in a complex matrix because of the specificity initially offered by the HPLC separation followed by hydrolysis from the selected bioreactor. Enzymatic hydrolysis prior to HPLC would result in hydrolysis of all peptides present in a sample, possibly to similar products, making a specific HPLC separation and MS determination of a single specific peptide difficult. The separation of a-endorphin, P-endorphin, and /I-endorphin fragment l-27 in CSF followed by tryptic hydrolysis and thermospray MS detection is shown in Fig. 7. The full scan spectra acquired contain the tryptic fragment pattern for each endorphin and a weak molecular ion due to partial hydrolysis when a 25% isopropanol aqueous solution is used. The spectra contain sufficient tryptic fragmentation to confirm the identity of the endorphins, as demonstrated by the mass spectrum of @-endorphin fragment l-27 in Fig. 7. The detection of lower molecular weight hydrolysis products should drastically improve sensitivity over the detection of the unhydrolyzed endorphin, as previously mentioned. Using selected ion monitoring for thermolysin fragments TL2 (m/z 443), together with post-

FIG. 7. HPLC/trypsin bioreactor/thermospray MS chromatogram for the analysis of a mixture of three endorphins (200 pmol of each) in cerebrospinal fluid. The separation was performed using 25% isopropanol in aqueous ammonium acetate (0.1 M) solution at a pH of 7.5 at a flow of 1.0 ml/min. Below the chromatograms is the thermospray spectrum of fl-endorphin fragment l-27 acquired from the analysis of the endorphin mixture in CSF.

IMMOBILIZED

ENZYME

BIOREACTORS

79

O-Endorphin (TLJ

01 1o:oo

I 20:oo

I 30:oo

40:oo

Time (minj

FIG. 8. HPLC/thermolysin bioreactor/thermospray MS chromatogram for thermolysin hydrolysis fragment TL2 from 800 fmol p-endorphin and 1.2 pmol of fl-endorphin fragment l-27 spiked into CSF. The chromatogram was generated using single ion monitoring (m/z 443) and post-HPLC column aqueous buffer addition to improve thermolysin activity.

HPLC column aqueous buffer addition, thermospray detection limits in the high femtomole range (700-900 fmol) for P-endorphin and /3-endorphin fragment l-27 can be achieved in a spiked biological matrix (Fig. 8). These detection limits are significantly better than the 30 pmol detection limits obtained upon the [M + 2H]‘+ ion of unhydrolyzed /3-endorphin (11). Furthermore, the sensitive detection of a endorphin can be based on the presence of several characteristic low mass hydrolysis fragment ions (i.e., TL1, TL2, TL8, TLg , TLlo, and TL,). This methodology offers superior specificity compared to detection based on one ion ([M + 2H]“) for thermospray HPLC/MS without enzymatic hydrolysis, or compared to radioimmunoassay. The HPLC/immobilized enzyme bioreactor/thermospray MS method could be used to validate and quantitate spiked /3-endorphin in biological fluids. The development of a suitable quantitative method employing thermospray MS requires the use of internal standards due to variability in ionization using a thermospray interface. The choice of an internal standard requires similarity in chemical and physical properties to the target peptide while exhibiting a different mass from the target peptide. The use of slightly modified peptides such as leu5-/3-endorphin could meet the requirements when stable isotope-incorporated internal standards are unavailable. Leu5-P-endorphin coeluted with /I-endorphin, permitting verification of conditions during analysis of the target peptide (P-endorphin), and tryptic fragment T1 is 18 Da lower (mw 1000) than T1 for fi-endorphin due to the substitution of leucine for the methionine. The HPLC/trypsin bioreactor/thermospray MS method could be used to generate a calibration curve based upon the area ratio of T1 or P-endorphin to T1 for a constant level (70 pmol) of internal standard (leu’-P-endorphin) from 3 to 170 pmol with a linear correlation coefficient

80

VOYKSNER,

CHEN,

AND

SWAISGOOD

Clearly, mass spectrometry is a detector that can be selective for a broad range of neuropeptides. Future work with combining microbore HPLC/immobilized enzyme bioreactors with electrospray MS can overcome some of the limitations in sensitivity, permitting femtomole full scan detection limits for most peptides (38,39). Tl

B-Endorphin

ACKNOWLEDGMENT This work Grant 5 ROl

Time

(mini

FIG. 9. HPLC/trypsin bioreactor/thermospray MS ion chromatograms from trypsin fragment T, (m/z 1001) for the internal standard leu5+endorphin (70 pmol) and fragment T, (m/z 1019) for /3-endorphin (10 pmol) spiked into CSF.

by the National

Institute

of Drug

Abuse,

REFERENCES 1 Swaisgood, H. E., and Catignani, G. L. (1987) Methods in Enzymology (Mosbach, K., and Andersson, Eds.), Vol135, pp. 596-604, Academic Press, San Diego.

2 Swaisgood, technology Publishing

of 0.9972 (n = 8). The analysis of a CSF sample spiked with 10 pmol of P-endorphin (Fig. 9) was measured to contain 12 pmol using this calibration. The ion chromatograms (Fig. 9) indicate that no interferences are observed for the detection of tryptic fragment Ti from pendorphin or the internal standard. The coelution and similarity between internal standard and sample precludes the use of other tryptic fragment ions for confirmation. The similarity between the measured and the spiked values indicates the HPLC/immobilized enzyme bioreactor/thermospray MS analysis could be used for the detection and quantitation of endorphins at the low picomole level. Quantifiable limits of detection could be slightly lower by basing the analysis of ,&endorphin and internal standard on chymotrypsin fragment C, (mw 573). The better sensitivity due to superior ionization transmission and detection efficiency for the lower mass fragments can improve the quantifiable detection limits from 800 fmol to 1 pmol. However, these lower molecular weight hydrolysis products are more prone to mass interferences from biological matrices (chemical noise) possibly requiring additional sample cleanup if routinely employed. HPLC/bioreactor/thermospray MS was very specific for the detection of added endorphins. The combination of HPLC retention time and the detection of several specific enzymatic hydrolysis products could be used to uniquely identify or verify a peptide. The detection limits of 800 fmol-1 pmol permits the monitoring of endogenous peptides in some areas of the central nervous system. Although radioimmunoassay (RIA) and radioreceptor assay methods can achieve better sensitivity (36), than the presented method, their molecular specificity and ability to identify unknown peptides can be limited to cross-reactivity for other peptides containing similar residues (37). Furthermore, the identification of a possible unknown peptide in the absence of standards is next to impossible based only on HPLC and RIA data.

was supported DA 04202.

H. E. (1985) Enzymes and Immobilized Cells in Bio(Laskin, A. I., Ed.), pp. l-24, Benjamin/Cummings Co., Menlo Park, CA.

3. Swaisgood,

H. E., and Horton, H. R. (1989) Immobilized Enzymes as Processing Aids or Analytical Tools, (Whitker, J. R., and Sonnet, P. E., Eds.), Vol 389, pp. 242-261, ACS Symposium Series, Washington D.C.

4. Bateman, A., Dell, A., and Morris, H. R. (1985) J. Appl. Biochem. 7,126-132. 5. Barber, M., Bordoli, R. S., Elliott, G. J., Sedgwick, R. D., and Tyler,

A. N. (1982)

Anal.

Chem.

54(4),

645A-657A.

6. Biemann, K., and Scoble, H. A. (1987) Science 237,992-998. 7. Tomer, K. B., Gross, M. L., Zappey, H., Fokkens, R. H., and Nibbering, N. M. M. (1988) Biomed. Environ. Mass Spectrom. 15, 649-657. 8. Blakley, C. R., Carmody, J. J., and Vestal, M. L. (1980) J. Amer. Chem. sot. 102,5931-5933. 9. Blakley, C. R., and Vestal, M. L. (1983) Anal. Chem. 55,750-754. 10. Rudewicz, P. J. (1988) Biomed. Environ. Mass Spectrom. 15,461463. 11. Voyksner, Spectrom. 12. Pilosof, Biomed.

R. D., and Pack,

T. P. (1989)

Biomed.

Environ.

Mass

l&897-903. D., Kim, H. Y., Vestal, M. L., and Dyckes, Mass Spectrom. 11,403-407.

13. Pilosof, D., Kim, H. Y., Dyckes, Anal. Chem. 56,1236-1240.

D. F., and Vestal,

14. Stachowiak, K., Wilder, C., Vestal, M. L., and Dyckes, J. Amer. Chem. Sot. 110,1758-1765.

D. F. (1984) M. L. (1984) D. F. (1988)

15. Kim, H. Y., Pilosof, D., Dyckes, D. F., and Vestal, M. L. (1984) J. Amer. Chem.Soc. 106,7304-7309. 16. Hearn, S. M., Regnier, F., and Wehr, C. (1983) High Performance Liquid Chromatography of Protein and Peptides, Academic Press, Orlando, FL. 17. Desiderio, D. M. (1984) Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry, Elsevier Science. Amsterdam. 18. Janolino, V. G., and Swaisgood, H. E. (1982) Biotechnol. Bioeng.

24,1069-1080. 19. Cuatrecasas, P. J. (1970) Biol. Chem. 245,3059-3065. 20. Duval, G., Swaisgood, H. E., and Horton, H. R. (1984) J. Appl. Biochem. 6,240-250. 21. Walsh, K. A. (1970) in Methods in Enzymology, (Perlmann, G. E., and Lorand, L., Eds.), Vol. 19, Academic Press, New York. 22. Taylor, J. B., and Swaisgood, H. E. (1980) Biotechnol. Bioeng. 22, 2617-2631.

DETECTION

OF

ENDORPHINS

USING

23. Worthington, C. C. (1988) Worthington Manual-Enzymes and Related Biochemicals, Worthington Biochemical Co., Freehold, NJ. 24. Folk, J. E., and Schirmer, E. W. (1963) Biol. Chem. 238, 38843894. 25. Folk, J. E., Piez, K. A., and Gladner, J. J. (1960) Biol. Chem. 235, 2212-2277. 26. Kuhn, R. W., Walsh, K. A., and Neurath, H. (1974) Biochemistry

13,3871-3877. 27. Hummel, 1399.

B. C. W. (1959)

28. Feder,

J. (1968)

29. Feder,

J., and Schuck,

30. Brown, 31. Houmard, 32. Yamada, 213-221.

Biochem.

J. Biochem.

Biophys. F. (1973)

Znt. J. Pept.

S., and Desiderio,

Physiol.

Res. Commun.

J. M. (1970)

W. E., and Wold, J. (1976)

Canad.

1393-

9,2784-2791.

Bioc!zemistry

12,828-834.

Anal.

Biochem.

81

BIOREACTORS

R. D., and Haney,

34. Robins, R. H., and Crow, trom. 2(2), 30-34. 35. Voyksner, R. D., Bursey, Chem. 56,1507-1514.

C. A. (1985)

F. W. (1988)

Rapid

J. T., and Pellizzari,

Anal.

Chem.

Commun.

57,

991-

MUSS Spec-

E. D. (1984)

Anal.

36. Hendren, R. W. (1986) Opioid Peptides: Molecular Pharmacology, Biosynthesis and Analysis, Nat. Inst. Drug Abuse Research Monograph Series No. 70 (R. Rapaka and R. Hawks, Eds.), pp. 255-303, Rockville, MD.

38. Whitehouse, (1985) Anal.

Res. 8, 199-204.

(1982)

33. Voyksner, 996.

ENZYME

37. Desiderio, D. M. (1984) Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry, pp. 93-113, Elsevier, Amsterdam.

32,326-332.

Biochemistry

Protein

D. M.

37,

IMMOBILIZED

127,

C. M., Dreyer, R. N., Yamashita, Chem. 57,675-679.

39. Loo, J. A., Udseth,

179,404-412.

H. R., and Smith,

M., and Fenn,

R. D. (1989)

Anal.

J. B.

Biochem.

thermospray mass spectrometry for the determination of neuropeptides.

Peptidases, including chymotrypsin, thermolysin, trypsin, V8 protease, and carboxypeptidases A, B, and Y, were immobilized for use in conjunction with...
1MB Sizes 0 Downloads 0 Views