Elucidation of the primary structures of proteins by mass spectrometry

ANALYTICAL

BIOCHEMISTRY

1%,118-124

(1991)

Elucidation of the Primary Structures by Mass Spectrometry Jean B. Smith,*

Gkaldine

Thkvenon-Emeric,*

David

of Proteins

L. Smith,*

*Department of Medicinal Chemistry and Pharmacognosy, Purdue University, Indiana 47907; and TVG Biotech, Altrincham, Cheshire, United Kingdom

Received

August

and Brian

Green?

West Lafayette,

1,199O

A combination of new mass spectrometric methods that can be used to determine the primary structures of proteins, including post-translational modifications, with unprecedented speed and accuracy is described. Structural characterization of cr-crystallins from bovine lenses has been used to illustrate the methods. The molecular weights of a-crystallins fractionated, but not to homogeneity, by reversed-phase HPLC were determined with an uncertainty of 0.01% which is at least 100 times more accurate than is possible using conventional methods. This information was used to identify the primary gene product as well as its phosphorylated and truncated forms. Molecular weight maps of proteolytic digests of these proteins were determined by directly coupled capillary HPLC fast atom bombardment-mass spectrometry. From these maps, the entire amino acid sequence was confirmed, and the phosphorylated peptide was identified. The MS/MS daughter ion mass spectrum of the phosphorylated peptide provided sufficient information to determine which residue was phosphorylated. Because protein structure, including post-translational modifications, is determined on the basis of molecular weight, this method has broad application and will be useful for a variety of diverse and challenging problems in protein structure elucidation. 0 1991 Academic Press, Inc.

Understanding the functions of a protein at the molecular level is greatly facilitated by knowing its complete primary structure, which includes the amino acid sequence as well as the type and sites of any post-translational modifications. With current methods for sequencing DNA, the amino acid sequences of large proteins can be predicted with relative ease and high accuracy. However, these approaches give no information regarding post-translational modifications. In ad-

dition, predicted sequences may not reflect the sequences that are actually expressed, either because of heterogeneity in the code or because of errors in sequencing the cDNA. Hence, there is a need for analytical methods that can be used to confirm predicted sequences and to identify and locate post-translational modifications. In addition, as methods for synthesizing long and complex peptides evolve, there is a concurrent need for techniques to characterize these unusual peptides. Most of the features of primary structure, amino acid sequence and post-translational modifications, are directly related to the molecular weight of either the intact protein or peptides derived from the protein. Fast atom bombardment-mass spectrometry (FAB-MS)’ has proved useful for identifying modified peptides (1,2). The success of FAB-MS may be attributed, in part, to the accuracy with which molecular weight may be determined (0.01%) and to the fact that analyses often can be performed with mixtures of peptides. FABMS has been most successful when used to analyze proteins with molecular weights less than 8000. Because of the molecular weight limitation of FAB-MS, its use for investigations of proteins has been limited to the analysis of peptides derived from the protein. Applications of FAB-MS to the analysis of proteolytic digests of proteins are complicated because some peptides may be transparent to FAB-MS, because cleavage of the protein cannot always be reliably predicted, and because the sample may contain other proteins as impurities. With the advent of electrospray-mass spectrometry (ES-MS) (3-5), the molecular weights of proteins can also be determined with an accuracy of 0.01%. This report illustrates how ES-MS can be used in con-

1 Abbreviations used: FAB-MS, fast spectrometry; ES-MS, electrospray-mass

atom bombardment-mass spectrometry.

118 All

Copyright 0 1991 rights of reproduction

0003-2697/91 $3.00 by Academic Press, Inc. in any form reserved.

MASS

SPECTROMETRIC

ELUCIDATION

junction with a new form of FAB-MS, capillary HPLC FAB-MS (6,7), as a new approach for identifying and locating post-translational modifications to protein. The power of these new mass spectrometric methods for investigating both unmodified and post-translationally modified protein structure will be demonstrated with a-crystallins (M, 20,000) isolated from bovine lenses. In this investigation, ES-MS was used to determine the molecular weights of the primary gene product, as well as phosphorylated or truncated forms of Lu-crystallins with an accuracy of 2 Da. Capillary HPLC FAB-MS was used to screen proteolytic digests of the proteins for modified peptides, which were subsequently analyzed by linked scan MS/MS to determine the exact site of modification. Using this procedure, we were able to unambiguously verify the sequence of LYA~, as determined by van der Ouderaa et al. (8), and to demonstrate that it is phosphorylated in vivo at Ser 122.

MATERIALS

AND

METHODS

OF

PROTEIN

119

STRUCTURE 3

--

I

I

I

c-

#H--

MC

c-

e--

c-

40

30

I T 20 .J m I

A

10 4

i

5 1,

6

0

Time (min)

FIG. 1. Reversed-phase HPLC separation of bovine lens a-crystallins. ES-MS analysis of the numbered peaks indicated that they are aA proteins. Results and tentative assignments of ES-MS analysis are given in Table 1.

Freehold, NJ) and chymotrypsin (Sigma Chemical Co., St. Louis, MO) were performed using 5 nmol of the proBovine lenses from 2-year-old cows were obtained tein and a 5O:l ratio of protein to enzyme in 200 ~1 of 0.1 from the Purdue University Animal Sciences Pilot M Tris, pH 8.2, at 37°C for 4 h. Approximately 100 pmol Plant and either processed immediately or stored at (0.5 ~1) of this digest, previously desalted, was injected -20°C. The lenses were homogenized in 0.5 M NaCl, onto a reversed-phase- HPLC column (Vydac Cl8 0.05 M Tris, 0.001 M EDTA, pH 7.4, for 1 h and centribonded silica, 5 km, 300 A, 150 X 0.3 mm id.), which was fuged at 15,000g for 1 h. Gel filtration chromatography coupled to a Kratos MS-50RF FAB mass spectrometer on Sephadex G-200 (Pharmacia, Piscataway, NJ) of the via a continuous-flow interface (12). A Rainin gradient soluble fraction gave three peaks corresponding to CY-, HPLC system was used to generate a flow of 70 /*l/min p-, and y-crystallins (9). The a-crystallin portion was which was split to deliver a flow of 3 pl/min to the colfractionated further by reversed-phase HPLC (Rainin umn. Injections (0.5 ~1) were made with a Rheodyne Instrument Co., Woburn, MA) using a Dynamax 250 valve (Model 7520). The entire effluent from the capilX lo-mm-i.d., 300-A, C4,5-pm column with a linear gralary HPLC column was directed to the continuous-flow dient of 20-60% CH,CN in H,O, with 0.1% trifluoroaceFAB probe. Solvents A and B contained 10 and 80% tic acid, over 40 min (10). Aliquots of these fractions acetonitrile, respectively. Both solvents contained 3% were analyzed by ES-MS or digested and analyzed by glycerol, 2% thioglycerol, and 0.1% trifluoroacetic acid. FAB-MS. The gradient was from 0 to 50% B. The mass spectromeIntact proteins were analyzed on a VG BIO-Q mass ter was operated at a scan rate of 30 s/decade and 8 kV spectrometer (VG Biotech, Manchester, UK) which em- accelerating voltage. Alternatively, portions of the diploys an electrostatic spray ion source operating at at- gest were collected after fractionation by reversedmospheric pressure and a quadrupole mass analyzer phase HPLC and analyzed on the same instrument by with an upper mass limit for singly charged ions of 3000. conventional FAB-MS techniques. MS/MS daughter Dried fractions were dissolved in water/methanol/aceion mass spectra were recorded by simultaneously scantic acid (49/49/l) and injected into the mass spectromening the electric and magnetic sectors. Helium was used ter at a rate of 5 pl/min. Mass spectra were recorded as the collision gas. over the mass-to-charge range of 700 to 1500 by scanning at a speed of 11 s/scan cycle. Several mass spectra were summed to improve the signal-to-noise ratio. Mass RESULTS scale calibration employed the multiply charged ions The chromatogram for reversed-phase fractionation from separate introduction of horse heart myoglobin, of the a-crystallins (Fig. 1) has approximately 10 peaks, AI, 16951.5 (11). which were presumed to be due to the two primary gene A portion of the protein in each fraction was digested products, aA (8) and (wB2 (14), and their various modiinto peptides for analysis by FAB-MS. Digestions of the fied forms. Results from previous investigations indiprotein with trypsin (Worthington Biochemical Corp., cated that likely modifications include acetylation of

120

t!,l

19.911.8 19.831.3 +16 +17

1 +I8

Jj 1100

1150

1200

1250

1300

1350

FIG. 2. Electrospray mass spectrum of Peak 3 from the HPLC fractionation of cu-crystallins (Fig. 1). Only peaks with 15 to 18 charges are shown. m/z is the mass to charge ratio.

the N-terminus (8,13), deamidation of various Gln and Asn residues (14-16), phosphorylation (17-20), and loss of portions of the C-terminus (l&16,21). All of the chromatographic peaks were collected and analyzed by ES-MS. Only the fractions labeled l-5 are discussed in this communication. A portion of a typical ES mass spectrum (Fraction 3) is given in Fig. 2, which shows peaks for ions of the proteins plus 15-18 protons. The molecular weights given are mean values derived from several multiply charged ions in the series. The precision of these molecular weight determinations is indicated by the standard deviation (0.5 Da) for multiple measurements. The accuracy of these molecular weight determinations is indicated by the fact that the determined values for peak 3 (19,831.3, 19,911.8, and 19,743.g) differ from the values calculated from the amino acid sequence of (YA~ by 0.7-1.5 Da. The ES-MS spectra for Fractions l-5 (Fig. 1) show that proteins with molecular weights close to 19,832 and 19,912 are present in Fractions 1-4. In addition, Fractions 1 and 3 have a M, of 19,746, and Fraction 4 has four additional components (Table 1). Based on the excellent correlation of determined and calculated molecular weights (Table l), the peak corresponding to M, 19,832 was assigned to aA which is monoacetylated. The molecular weight of the other major component (19,912) is 80 Da higher, suggesting that it is a monophosphorylated or sulfated form of aA2. The minor component with M, 19,745 corresponds to a degradation product of arA2 which has lost the C-terminal residue,

AL.

Ser 173, and is designated CXA~~-‘~~.The molecular weights of the other proteins in Fraction 4 are less than that of aA2, suggesting that they may be degradation products. These proteins were identified through a computer-assisted analysis procedure (22) which was used to systematically search the sequence of (uA2 for all possible segments that would have a molecular weight corresponding to the experimentally determined values. Possible sequences considered included aA2, N-terminal acetylated aA2, cuAl ((uA2 phosphorylated at Ser 122 as proposed by Voorter et al. (18)), and acetylated cuAl. The assignments listed in Table 1 are the possibilities for aA that has been hydrolyzed once. Directly coupled HPLC FAB-MS with a capillary HPLC column is the preferred method for producing molecular weight maps of digests because the narrow diameter of the column gives a small elution volume and an excellent recovery for subnanomole amounts of material. This technique was applied to tryptic and chymotryptic digests of Fraction 3 to locate the site of phosphorylation (or sulfation) on aA2. Results from these analyses are summarized in Fig. 3, where T and C denote tryptic and chymotryptic peptides, which were identified by their molecular weights. These results confirm that all except two amino acids of the proposed sequence of otA2 are correct, and that the N-terminus is acetylated. Assignment of Ser and Cys to the two remaining amino acids (in either order) is consistent with the molecular weight of the protein determined by ES-MS. To determine the site of phosphorylation (or sulfation), the molecular weight map of the chymotryptic digest was searched for a pair of peptides with molecular weights differing by 80 Da. The FAB mass spectrum shown in Fig. 4 has such a pair of peptides, MH+ 1199 and 1279, corresponding to the normal and modified 119-129 segment of crA2. The peak at m/z 758 corresponds to chymotryptic peptide 75-80 (Fig. 3). No evidence for modifications at other sites of aA2, including deamidation of Asn or Gln, was found. These results demonstrate that one of the residues in the 119-129 segment is modified. Complete identification and exact location of the modification were possible from peaks in the daughter ion MS/MS spectrum of the peak at m/z 1279 (Fig. 5). The large peak due to a loss of 98 (H,PO,) identified the modification as phosphorylation (23). Sulfation would have been indicated by a loss of 82 (H,SO,) (23). The possible sites of phosphorylation in the chymotryptic peptide 119-129 are Ser 122 and Ser 127. The peaks in the MS/MS spectrum of the phosphorylated peptide showing the C, fragment at m/z 549 (Fig. 5) and other fragments of the A, B, and C series (24) with +80, as well as the YE fragment at m/z 1010 with +80, establish the site of phosphorylation as Ser 122.

MASS

SPECTROMETRIC

ELUCIDATION

OF

TABLE

PROTEIN

121

STRUCTURE

I

Molecular Weights and Assignments of aA-Crystallins Fractionated by Reversed-Phase and Analyzed by Electrospray-Mass Spectrometry ES-MS Peak

lb

Peak

19,831.8 19,911.7 19,746.7

molecular

2

Peak

19,831.7 19,911.g

weight 3

Peak

19,831.3 19,911.8 19,743.g

4

Peak

19,832.6 19,913.5 17,570.l 17,651.3 11,990.o 17,915.8

D Average molecular weight. * Peak number refers to Fig. 1. c Read as “acetylated oA2, residues d Another possibility is N-terminally e Another possibility is N-terminally

l-173.” degraded degraded

olA1 20-173 &Al 68-173

(M, (M,

11,992.3

The analysis of large biopolymers with mass spectrometers of limited mass-to-charge range is possible not only because sample introduction by electrospray solves the volatization problem, but also because it produces ions with multiple charges. As a result, mass spectrometers with an upper m/z limit of 2000 can be used to detect multiply charged ions with molecular weights greater than 2000. For example, the multiply charged molecular ions of bovine albumin dimer (I& 135,000) can be produced by ES-MS (3). ES-MS is also attractive because sample introduction requirements closely parallel conditions normally used for reversed-phase HPLC separation of proteins. Proteins are dissolved in a mixture of water, acetonitrile, and trifluoroacetic acid

(1089.5)

(587.3)

TT-T81Cll

VQEDPVBIHGKHNB ---+?I.6) (1223.6) -Tll-,-T12-T13-

130

(641.4)

(1017.5)

SCSLSADGMl%SGPKIF’SdtGHSERA&SRBBKI’S&’SS

&347-

YJ28)

(491.2)

FIG. 3. chymotryptic

Peptides found by FAB-MS (C) digests of Peak 3 (Fig.

analysis 1).

of tryptic

(T)

5

Assignment AC-crA2 AC-cuA1 AC-oA2 AC-oA2 AC-cuA1 AC-aA Ac-oAl

I-173’ l-173 1-172 l-151d l-151 l-101’ 1-154

Calculated

Mt

19,832.5 19,912.5 19.745.4 17,571.0 17.651.0 11,991.8 17,916.3

17,570.8). 11,988.5).

DISCUSSION

(1299.6)

HPLC

and

and introduced into the mass spectrometer via a capillary tube which is at a potential several kilovolts away from ground and terminates at atmospheric pressure. The electrical field at the tip of the capillary charges the surface of the liquid, dispersing it by coulombic forces into a fine spray of charged droplets. Solvent evaporation, assisted indirectly by the coulombic force due to the high concentration of ions within the droplet, gives rise to molecular ions of the protein. The ES mass spectra of proteins have a series of peaks, each corresponding to the neutral molecule plus some number of excess protons. The number of excess protons, which is not the same for all of the molecules, is related to the number of basic residues (Lys and Arg). The molecular weight of the protein is deduced from the mass-to-charge ratios of adjacent peaks, which differ in mass and charge by one proton. The ES mass spectrum of a mixture of proteins has a series of peaks for each component, as illustrated for aA and phosphorylated aA (aAl) in Fig. 2. The significance of ES-MS to the field of protein chemistry may be appreciated by comparing typical values for accuracy, resolution, and required sample size of ES-MS with those of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Table 2). The molecular weights of proteins can be determined by ESMS with an uncertainty of less than a few mass units (O.Ol%), which is sufficient to distinguish among many of the common post-translational modifications, such as oxidation, acetylation, and phosphorylation. Deamidation increases the molecular weight by 1 Da, and is an example of a post-translational modification that could not be detected by ES-MS at its present level of development. Molecular weights determined by SDS-PAGE may have large errors since it is the hydrodynamic volume of a protein that is measured and not its mass. Although the error in molecular weight determination

122

SMITH

ET

AL.

0

700

800

900

1000

1100

1200

1300

m/z

FIG. 4. A scan from the on-line 1199) and nA1 (MH+ 1279). The

capillary HPLC FAB-MS peak at m/z 758 is peptide

analysis 75-80.

of a chymotryptic

by SDS-PAGE is often about l%, errors are frequently greater than 10%. ES-MS also offers improved resolution for analysis of protein mixtures. For the quadrupole mass spectrometer used in this study, proteins with molecular weights differing by as little as 0.1% would give discernible peaks. This resolution is about a factor of 10 better than that with SDS-PAGE. Given the early stage of development of ES-MS, neither the ultimate mass resolution nor the limit of detection has been established. However, at this time, ES-MS is approximately as sensitive as SDS-PAGE when used with Coomassie blue staining. An important role for ES-MS in detecting and identifying proteins has been illustrated for aA-crystallin. The amino acid sequence of aA was determined by van der Ouderaa et al. (8), who used a combination of manual and automated methods to sequence peptides in proteolytic digests of (YAP. The average molecular weight of aA with an acetylated N-terminus, based on their sequence, is 19,832.5, which differs from the value determined by ES-MS by 1.2 Da. Since the most likely errors in sequenation (missed residues, incorrect assignments) result in changes in molecular weight greater than 1 mass unit, this excellent correlation strongly corroborates the reported sequence. Early investigators noted several proteins in the electrophoretic pattern of aA. At that time it was proposed that the major proteins were aA and its deamidation product, c~A1, with Asn 123 as the probable deamidation site (14-16). Subsequent studies showed that arA1 is (uA2 that has been phosphorylated at Ser 122 (17-20). The other proteins were believed to be degradation products of cuAl and aA2, i.e., ~yA’-“l, aA1-151, aA1-lW, and ,A1-16’ (1521). Our ES-MS data (Table 1) showing molecular weights consistent with N-terminally acetylated a-crystallins degraded from the C-terminus to form aA’-‘o’ (M, 11,990), aA1’-‘51 (17,651), and c~A2l-l~~

digest

of Peak

3 showing

peptide

119-129

of (uA2 (MH+

(17,571) support some of these early assignments for degradation products and are consistent with aAl being phosphorylated aA2. Although the molecular weight found for (YA~ strongly supports the proposed amino acid sequence, the matching molecular weight does not exclude the possibility that peptides derived from cuA2 were assembled incorrectly. If such errors had been made in the original sequence determination, it would be apparent from the molecular weight maps of peptides in proteolytic digests. Such maps are most easily determined through directly coupled capillary HPLC FAB-MS. Since some of the peptides in a digest may be transparent to FABMS, either because their molecular weights are too high or too low or because they are too hydrophilic, it may be necessary to analyze digests made with different enzymes to obtain a complete map. For (YAP, 77% was detected via a tryptic digest; 82% was detected as the chymotryptic digestion. As indicated in Fig. 3, the combined molecular weight maps of these digests covered 99% of the sequence of aA2. Similar information was also obtained by conventional FAB-MS analysis where HPLC fractionation and mass spectrometric analysis were performed in separate steps. On-line HPLC FAB-MS is attractive because it is fast and because the detection limit for low-molecular-weight peptides is improved by approximately a factor of 10. The inevitable sample losses incurred when fractions are collected, dried, and reconstituted for analysis by conventional FAB-MS analysis are circumvented with on-line HPLC FAB-MS. The ability to analyze peptides in mixtures has been a major advantage of FAB-MS. However, when current methods are used to analyze complex mixtures typical of proteolytic digests, many peptides are not detected and a complete molecular weight map is rarely achieved. For example, only 48% of cuA2 was found when the un-

MASS

SPECTROMETRIC

ELUCIDATION

OF

PROTEIN

123

STRUCTURE

1279.6

amino acid

R5

R6

R7

Rs

(N)

VI

CD)

(0)

(3

R9

ion of the 119-129 ion.

peptide

%O (4

RI1 (6

Y series

FIG. 5. E/B in the daughter

linked scan daughter ion spectrum with

ion mass spectrum specific fragments

of the molecular of the molecular

fractionated tryptic digest of aA was analyzed by direct injection continuous-flow FAB-MS. A similar result was obtained when the unfractionated digest was analyzed by normal FAB-MS. From this failure it was apparent that fractionation by reversed-phase HPLC

TABLE

2

Comparison of Accuracy, Resolution, and Sensitivity Electrospray-Mass Spectrometry and SDS-PAGE ES-MS Accuracy Resolution Sample size

0.01% (2/20,000) 0.1% (20/20,000) l-10 pm01

“For Coomassie blue staining sensitivity is possible with other

of

SDS-PAGE l-20% 1% 5-50 pmol”

of a protein of iW, 20,000. staining methods.

Higher

of cyA1 (MH*

1279).

The inset

relates

peaks

prior to analysis by FAB-MS was needed to obtain complete molecular weight maps. On-line capillary HPLC continuous-flow FAB-MS offers a practical solution giving fractionation, rapid analysis, and high sensitivity in one step. A complete peptide map can be obtained in less than 1 h. The results presented here demonstrate that the primary structures of proteins can be investigated by mass spectrometry with accuracy and speed unmatched by any other analytical technique. This study of a-crystallins has shown that ES-MS can be used for determining the molecular weights of intact proteins with unprecedented accuracy, even when proteins are present as a mixture. This information can be used to identify the protein and to indicate which types of modifications are likely. On-line capillary HPLC FAB-MS can be used to locate sites of modifications to specific segments of the protein which are identified by their molecular weights.

124

SMITH

The structure elucidation problem can be subdivided further by recording the gas-phase fragmentation of the molecular ions of peptides by MS/MS. The amino acid sequences of peptides, as well as the types and sites of modification, are determined by reassembling the fragment ions using established rules of peptide fragmentation. ACKNOWLEDGMENTS This investigation was supported by Grants GM R0140384 ROl 07609 from the National Institutes of Health.

and EY

1. Desiderio, D. (Ed.) (1990) Mass Spectrometry Press Reviews, CRC Press, Boca Raton, LA. J. A. (Ed.) (1990) Press, San Diego.

3. Covey, T. R., Bonner, Rap. Commun. Mass

Methods

5. Loo, J. A., Edmonds, 201-204.

of Peptides,

in Enzymology,

C. G., and Smith,

193,

J. (1988)

10. Perry, R. E., and Abraham, E. C. (1986) J. Chromatogr. 103-110. 11. Evans, V., and Brayer, G. D. (1988) J. Biol. Chem. 263, 4268. 12. Caprioli, bardment

R. M. Mass

(Ed.) (1990) Spectrometry,

Continuous-Flow Wiley, Sussex.

6. Ito, Y., Takeuchi, T., Ishii, D., and Gato, M. (1985) 346,161-166. 7. Caprioli, R. M., DaGue, B., Fan, T., and Moore, Biochem. Biophys. Res. Commun. 146,291-299. and

Science

248,

J. Chromatogr. W. T. Bloemendal,

F. A. M., Koopmans, M., van Venrooij, H. (1979) Exp. Eye Res. 28,223-228.

(1987) H.

W. J., and

Fast

Atom

361, 4263Bom-

13. van der Ouderaa, F. J., de Jong, W. W., Hilderink, mendal, H. (1974) Eur. J. Biochem. 49, 157-168.

A., and Bloe-

14. Bloemendal, H., Berns, A. J. M., van der Ouderaa, Jong, W. W. (1972) Exp. Eye Res. 14,80-81.

F. J., and de-

15. van Kleef, F. S., de Jong, W. W., Nature (London) 258, 264-266.

H. J. (1975)

and

Hoenders, W. W.,

17. Spector, A., Chiesa, R., Sredy, J., and Garner, Natl. Acad. Sci. USA 82,4712-4716. 18. Voorter, C. E. M., Jong, W. W. (1986)

19. Chiesa, R., Gawinowics-Kolks, Biol. Chem. 262,1438-1441.

23. Gibson, B. W., Poulter, Enzymology (McCloskey, San Diego.

Proc.

H., and de A. (1987)

J.

N. J., and Spec-

and Bloemendal,

22. Smith, D. L., and Zhou, Z. (1990) in Methods (McCloskey, J. A., Ed.), Vol. 193, pp. 374-389, San Diego.

Hoenders,

W. (1985)

M. A., and Spector,

21. de Jong, W. W., van Kleef, F. S. M., Eur. J. Biochem. 48, 271-276.

24. Roepstorff, 11,601.

and

Mulders, J. W. M., Bloemendal, Eur. J. Biochem. 160,203-210.

20. Chiesa, R., Gawinowics-Kolks, M. A., Kleiman, tor, A. (1988) Exp. Eye Res. 46, 199-208.

S. F., and Whitehouse,

R. D. (1989)

8. van der Ouderaa, F. J., de Jong, W. W., (1973) Eur. J. Biochem. 39, 207-222.

CRC

Vol.

R. F., Shushan, B. I., and Henion, Spectrom. 2, 249-256.

4. Fenn, J. B., Mann, M., Meng, C. K., Wong, C. M. (1989) Science 246,64-71.

9. Asselbergs, Bloemendal,

AL.

16. van Kleef, F. S. M., Willems-Thijssen, H. J. (1976) Eur. J. Biochem. 66,477-483.

REFERENCES

2. McCloskey, Academic

ET

H. (1974)

in Enzymology Academic Press,

L., and Cohen, P. (1990) in Methods in J. A., Ed.), Vol. 193, Academic Press,

P., and Fohlman,

J. (1984)

Biomed.

Mass.

Spectrom.