- Email: [email protected]
New tools for protein sequence analysis
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Reply from Blalock I entirely agree with J. Slootstra and E. Roubos that 'receptor and hormone may have originated from a single DNA molecule in which one strand coded for the hormone and the other for the receptor'. In fact, our original article stated this 1. Similarly, we concur 2 with R. Brentani that co-evolution of interacting peptides coded for by comp-
lementary codons would speed up evolution. I do, however, disagree with his contention that the X-ray crystallography and site-specific mutagenesis work was misquoted. These techniques led to identification of the same sites of interaction of interleukin-2 and its receptor as would be predicted by the Molecular Recognition Theory.
New tools for protein sequence analysis Fumio Sakiyama Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation w h i c h facilitate determination of protein primary structure have recently been developed. The functional role of a protein depends on its specific threedimensional structure which is determined primarily by its amino acid sequence (primary structure). Protein sequence analysis is thus an essential step towards an understanding of a protein's function. Since the amino acid sequence is dictated by the nucleotide sequence, DNA sequencing (now a rapid, straightforward technique, relatively economical in lerms of materials required) has become a popular method of obtaining information on amino acid sequence, especially for large proteins of which only small amounts are a v a i l a b l e for study. However, this approach has the drawback that it provides no information on the structure of proteins modified post-translationally. Direct sequencing of the polypeptide chain(s) is therefore a preF. Sakiyama is at the Institute for Protein Research, Osaka University, Saita, Osaka 565, Japan.
ferred option when only nanomolar amounts of highly purified protein of molecular mass of 30 kDa or less are available. Recently, new instrumentation and improved techniques have been developed I 6. We discuss those which are now being used routinely as well as those which will become of practical importance in the near future. The strategy for protein sequence analysis has remained essentially unchanged for the past 30 years, and employs a series of reactions termed the Edman degradation. I n this, phenylisothiocyanate is reacted with the terminal amino group of a protein/peptide yielding a derivative which may be cleaved from the peptide chain. The cleaved residue undergoes a rearrangement to a phenylthiohydantoin (PTH) derivative of the amino acid, easily identified by HPLC. Repetition of this sequence of reactions permits the sequencing of the complete polypeptide, starting from the N-terminal
1990, Elsevier Science Publishers Ltd (UK) 0167- 9430/90/$2.00
References 1 Bost, K. L., Smith, E. M. and Blalock, J. E. (1985) Proc. Natl Acad. Sci. USA 82, 1372-1375 2 Blalock, J. E. and Bost, K. L. (1988) Recent Prog. Horm. Res. 44, 199-222 J. E D W I N B L A L O C K
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
end. Nowadays the majority of direct sequencing by Edman degradation is carried out in sequenators with most of the process automated.
Protein sequence analysis procedure A typical protein sequence analysis procedure consists of eight steps: (1) N-terminal analysis; (2) amino acid analysis; (3) protein fragmentation; (4) peptide fractionation; (5) Edman degradation and PTH-amino acid analysis; (6) C-terminal analysis; (7) location of the disulfide bond; and (8) determination of the mode and site of post translational modification. Step 1: N-terminal analysis This can be u s e d not only to determine the amino acid sequence of the N-terminal region but also to test the purity of the protein sample. A gas-phase or pulsed-liquid sequencer (see Glossary~ equipped with PTH-amino acid analyser can determine the 30-40 N-terminal amino acids by direct sequencing of 50-100 pmol of a protein. Under optimal running conditions, up to 10 amino acid residues may be determined from 5-10 pmol of a protein. The N-terminal sequencing of a protein which has been separated by SDS-PAGE and blotted to a polyvinylidenedifluoride (PVDF) (see Glossary) membrane is routine 7. Frequently, in the direct sequence analysis, a PTH-amino acid cannot be detected in the first cycle of Edman degradation of a protein. In most cases, this is due to the reaction being blocked by an acyl group at the N-terminus. The presence of an acetyl group at the N-terminus is a
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283
~Fig. mGIossary Polyvinylidenedifluoride (PVDF) m e m b r a n e - teflon-like m a t e r i a l n o t d a m a g e d by reagents and s o l v e n t s used in c o m m e r c i a l automated sequencing instruments. Proteins bind to the m e m b r a n e t h r o u g h h y d r o p h o b i c interactions and can be visualized by staining w i t h dyes. C o n t a m i n a n t s can be w a s h e d f r o m the m e m b r a n e by rinsing w i t h w a t e r . P u l s e d liquid (or pulse liquid) protein s e q u e n c e r - indicates that a m i n u t e (but quantified) a m o u n t of a reagent in liquid is s u p p l i e d like a pulse to the m e d i u m of E d m a n reaction. Before the i n t r o d u c t i o n of this m e t h o d the reagent w a s s u p p l i e d , u n q u a n t i t a t i v e l y , as a gas (gas-phase protein sequencer).
1 E, R2 i I ECO-NH-CH-CO-NH-CtI-CO- - -
R2
I I RCO-NH-CII-CO-N!t-CH-CO- - -
-Lys + NH~ - - -Lys + Nil- -
1
Phenylisothiocyanate
R, E2 I I RCO-NH-C!t-CO-NH-CH-CO. . . .
PTC PTC I I Lys 4 PTC-NH- - - L y s + PTC-NH- -
Performic acid oxidation
R, R2 I 1 RCO-Nt-gtt-CO-NH-CIf-go . . . .
PC I l,ys +
PC I PC-Nit- - - g y s + PC-NH- -
A A R E digestion
RCO-NtI-CH-COOit
Step 2: Amino acid analysis Amino acid analysis is essential to determine the composition of the intact protein and its peptide fragments. Precision has priority over sensitivity in routine amino acid analysis and the established ninhydrin method is well-suited for precise analysis, but the amount of protein (/> 100 pmol) necessary for quantitative analysis by this method
Lys-
API digestion
RI
E,
common modification of intracellular proteins. The acetyl group can be removed with acylamino acid-releasing enzyme (AARE) as acetylamino acid. However, AARE attacks only N~-acylated peptides of less than 40 residues and the N-acety]ated protein must be fragmented into peptides prior to enzyme digestion. Recently, we devised a procedure for removing the N-terminal acetylamino acid without isolation of the acylated peptide 8 (see Fig. 1). The same principle may be applied for removal of the N-terminal pyroglutamyl group, using pyroglutamylaminopeptidase instead of AARE. Confirmation of the chemically determined sequence of the N~-acylated peptide by mass spectrometry using soft ionization (see Box), mainly fast atom bombardment (FAB) and plasma desorption (PD), has become routine 9.
-I,ys. . . . .
Identification of N-acetylamino acid
+
122 I NH~-CH-CO. . . .
Pg I Lys
+
PC I PC-NH- - -l,ys + PC-Nil- -
A m i n o acid s e q u e n c e analysis
The N-terminal sequence analysis of an N
is large compared with the amounts required for protein sequence analysis. Optimization of the ninhydrin method* has enabled us to determine the amino acid composition (except for Pro) of a protein within + 5% for 50 pmol levels (Fig. 2) l°. Several post- and pre-column derivatization methods (Table 1) are used for high-sensitivity analysis at picomolar methods where error often increases beyond 10%. At least 10 pmol of the protein/peptide sample (including samples obtained by on-the-membrane vapor-phase hydrolysis; i.e. hydrolysis of a pro-
*The sensitivity of the amino acid analysis was optimized by modifying two important reaction steps. One was to increase the signal-to-noise ratio by optimizing the amino acid-ninhydrin colorization reaction and the other was to use gradient elution of amino acids instead of step elution to ensure baseline separation of each amino acid.
tein/peptide blotted on a membrane [such as PVDF] with gaseous hydrochloric acid) 11 is needed to give reproducible and reasonably accurate determinations in most of the high-sensitivity methods though their limit of detection is less than a tenth of the values shown in Table 1. The detection limit of amino acids may now be as low as the femtomole or attomole level for some methods; for example analysis using a set of capillary zone electrophoresis (CZE) and 4-dimethyl-aminoazobenzene-4'sulfonyl amino acids 12 or a set of CZE or open tubular liquid chromatography (OTLC) and laser-induced fluorescence detection of isoindole compounds that had been formed upon reaction with naphthalene-l,2dialdehyde of amino acids 13. In OTLC, the separation takes place inside a capillary tube (inner diameter of 1-50 gm; length I> 1 m), the stationary phase being attached
284
TIBTECH- OCTOBER 1990 [Vol. 8]
--Fig. 2 440nm
to the inner wall of the tube (instead of packed particles in the case of the conventional HPLC). OTLC has an advantage over CZE in that the separated material can be collected and used in subsequent experiments. However, in high sensitivity quantitative analysis using picomolar (or lower) amounts, a serious problem exists: how can one collect reliable data for the 'clean' sample, free from contamination by glycine, serine and their derivatives in the course of the purification and hydrolysis of a protein/peptide. Even deionized, distilled water is a possible source of contamination at these levels. In addition to the vapor-phase acid hydrolysis, the liquidphase hydrolysis of protein/peptide in a minimal amount of hydrochloric acid in sealed capilary tubes 14 may help to decrease this unfavorable amino acid contamination.
Step 3: Fragmentation Two proteases Achromobacter protease I (API or lysylendopeptidase) and Asp-N, each of which recognizes specific amino acid residues, have recently been introduced and used successfully in the fragmentation of proteins and peptides. API is a serine protease hydrolysing lysyl peptide bonds, including the Lys-Pro bond 15 and the S-aminoethylcysteinyl bond 16. Characteristics such as its proteolytic activity (greater than trypsin), stability in the presence of 5 M urea and 0.1% SDS, and wide pH optimum (pH 8.5-10.7) make it favored as an enzyme for protein fragmentation. API is also active at pH 6.5 where thio[-disulfide exchange is suppressed. API (consisting of 268 residues) 17, is more active and more stable than the endoproteinase Lys-C and is routinely used for the primary fragmentation of a protein in our laboratory. To prepare sufficient peptide fragments to cover the entire peptide chain, a combination of API and Staphylococcus aureus V8 protease digestion is used. Asp-N, a bacterial metalloprotease, hydrolyses the peptide bond at the N-terminal side of aspartic acid and cysteic acid 18 and is suited for subfragmentation of large peptides. Proteases and chemical reagents of practical importance in protein/ peptide fragmentation are listed in Table 2.
lOOpmol
o~
z
o~
~ ~ ru
m 0
~ ._,z,
~>i
~3_
co
~
dAd~ 2
"
(N
COCO(r; LO
~
I}
CO
~
50pmol
"
('0@
~ j ~
~.(D
OOOtO
C~• I ¢q
I.*O C'4
03
r--
r--
03 O~CO" " t'N ,'~ O-) C0
1
]
r
I
~
i
q
I
I
I
10
15
20
25
30
35
40
45
50
55
Time (min)
Chromatograms of 16 protein amino acids recorded with a Hitachi L8500S amino acid analyser by the ninhydrin method (50 and 100 pmol sample for each amino acid). Ordinate is absorbance at 570 nm. Inset on the upper chromatogram is for proline (200 pmol, 12.61 min) detected at 440 nm.
--Table I Typical methods for amino acid analysis by pre- and post-column derivatization A m i n o acid
derivatives or
Derivatization Detection A p p r o x i m a t e l o w e r (nm) limit of quantitation
reagents Ninhydrin PhenylthiocarbamoylA A b (PTC) 4-Dimethylaminoazobenzene-4'-sulfonylA A (Dabsyl) o-Phthaldehyde + RSH (OPA) (fluorescent) 9-FluorenylmethyloxycarbonyI-AA (Fmoc) (fluorescent)
(pmol) a post pre
570 254
50 10
pre
436
10
pre post pre
ex. 340 em.455 ex. 264 em.340
aValues are for routine analysis. bAbbreviations: AA, amino acids; ex., excitation; em., emission.
5-10 10
TIBTECH- OCTOBER1990[Vol.8]
285
- - T a b l e 2, Methods for the cleavage o f the peptide chain Proteases
Scissile bonds
Proteases/ reagents
Scissile bonds
API Lys-X Subtilisin nonspecific (Lysylendopeptidase)a Endoproteinase Lys-C Papain Lys-X nonspecific Trypsin Pepsin Arg/Lys-X nonspecific Arginylendopeptidase Arg-X Clostripain Arg-X BrCN Met-X V8 proteaseb Glu-X H+/H20 Asp-Pro Asp-N X-Asp Iodosobenzoic acid Trp-X Prolylendopeptidase Pro-X Asn-Gly NH2OH Thermolysin X-Leu/Val/lle/Met NTCBc or DMAPX-Cys CNd Chymotrypsin Tyr/Tr p/Phe/Leu-X X-Asp/Asn-Y H+/H20,heat aAvailablefrom Wako PureChemical Industries bStaphylococcusaureus C2-nitro-5-cyanothiobenzoicacid 1-cyano-4-dimthylaminopyridiniumperchlorate
Step 4: Peptide fractionation Before reverse-phase HPLC was introduced, peptide purification was a time-consuming and complicated part of protein sequencing. Although the use of HPLC saves time and effort in peptide purification, the recovery of peptides from a reverse phase column is in the range of only 3080%. In particular, hydrophobic peptides are often not eluted (or only with difficulty) from the column. To detect a possible missing peptide(s), FAB mass spectrometry is useful since hydrophobic peptides of 5 kDa and less are more easily dispersed than hydrophilic ones. Conversely, hydrophilic peptides adsorbed on a nitrocellulose membrane tend to desorb more easily than hydrophobic ones by 252Cf PD 19. Proteolysis of a protein on the probe tip of a FAB mass spectrometer saves time and sample 2o. In any event, a comparison of two 'mass summaries' obtained before and after reverse phase HPLC of the proteolytic digest of a protein is recommended to detect possible missing peptides 21. Another technique complimentary to reverse phase HPLC for peptide mapping is CZE 22. As little as 50 ng of protein is digested with trypsin, immobilized in a packed column and mapped by CZE 2a. Attempts have been made to construct the online mass spectral analysis system of a peptide mixture. When the formation of large fragments is anticipated in protein fragmentation, a combination of electrospray ionization mass spectrometry 24 and CZE or OTLC seems promising, since, in electrospray ionization, a protein
taining SDS at pH 3.11. The limit of detection of PTH-amino acids is as little as 500 fmol with a conventional HPLC apparatus. A more sensitive method has also been reported for the determination of the N-terminal amino acid released by Edman degradation. This method utilizes the ring-opening reaction of anilinothiazolinone by 4-aminofluorescein to a fluorescent phenylthiocarbamoylamino acid amide, which is detected at the attomolar level 26.
Step 6: C-terminal analysis
Few good methods for C-terminal analysis exist; hydrazinolysis and carboxypeptidase Y digestion are the only practicable means. Hydrazinolysis, which results in cleavage and sprayed from the end of a capillary derivatization to amino acid hydracolumn to a high electric field zine (with all except the C-terminal (3-4 kV) becomes a group of mul- residue being unmodified) has retiple charged molecules which can cently been improved as vaporbe detected in the limited range of phase hydrazinolysis. As a result, small mass-to-charge ratio (m/z). the duration of reaction has been Accordingly, this separation- shortened to 2-4 h, the detection detection method detects all pep- sensitivity improved and the recovery tide fragments including large pep- of labile amino acids such as Ser, tides which might be missed by FAB Thr and Asp increased 27. Carboxymass spectrometry. peptidase Y, unlike other carboxypeptidases which show some speciStep 5: Edman degradation and ficity in cleavage, will cleave any PTH-amino acid analysis C-terminal residue and may be used Protein sequencing is usually per- to identify the first few C terminal formed using a gas-phase or pulsed- residues sequentially. liquid sequencer with an on-line Three n e w methods for C-terminal PTH-amino acid analysis system. analysis have been reported. The Unlike solid-phase sequenators first one uses acid hydrolysis in where the polypeptide is coupled H21ao; only the C-terminal amino through its C-terminus to a support- acid remains isotopically unlabeled, ing matrix, in gas-phase sequencing and is easily distinguished by mass the polypeptide is strongly adsorbed spectrometry from the internal and on a glass fiber or PVDF paper, and N-terminal amino acids of a prothe Edman chemistry is performed tein 28. The two other methods inusing trimethylamine and trifluoro- volve the isolation of the C-terminal acetic acid in vapor phase, permit- peptide by means of specific adsorpting easier handling and reduced tion of the other peptides to an contamination. Although auto- appropriate solid support. One supmation of the Edman reaction for port is a dehydrotrypsin-liganded sequencing is well established, diffi- column which has no affinity for the culties such as the wash-out of part C-terminal peptide lacking Arg and of hydrophobic peptides, the partial Lys in the tryptic digest of a prorandom hydrolysis of peptide bond tein 29. (When the C-terminus of a with trifluoroacetic acid and the o~,[3- protein is Arg or Lys, the affinity rearrangement of the Asn-Gly pep- column also adsorbs the C-terminal tide bond still remain. Since the peptide and this method cannot be number of amino acids determined used directly for C-terminal analysis.) and the reliability of the result of The other support is p-phenylenesequencing depend mainly on the diisothiocyanate (DITC)-activated sensitivity and the reproducibility of glass, to which peptides in the APIPTH-amino acid analysis, we have digest of a protein are first bound devised 25 an isocratic elution analy- covalently and then the C-terminal sis recycling a formate buffer con- peptide is selectively released by
286
TIBTECH - OCTOBER 1990 [Vol. 8]
--Box I Soft ionization mass spectrometry techniques
one cycle of Edman degradation 3°. This works well when Lys is absent from the C-terminus. Efforts have also been made to improve the thiohydantoin method, which releases the C-terminus of a protein as amino acid thiohydantoin via the formation of a mixed anhydride of the C-terminal carboxyl group with isothiocyanic acid. At present, this is a sole potential candidate for the C-terminal Edmantype sequencing. However, the method has not grown to become routine for microanalysis at picomolar amounts 31 mainly because of its low efficiency of performance relative to Edman degradation in terms of sensitivity and the number of determinable amino acid residues.
Step 7: Location of disulfide bonds The determination of disulfide bonds in a protein using chemical methods is time-consuming and often requires nanomolar amounts of sample. Mass spectrometry is the prefered method for the rapid determination of disulfide bonds in a peptide whose amino acid sequence is known; the technique is based on the detection of a peak(s) whose m/z value changes before and after the reduction with 1,4-dithiothreitol or the performic acid oxidation of disulfide bonds 32. The on-the-probe cleavage of disulfide bonds enables the analysis to be very sensitive, and the location of disulfide bonds can be determined using just 100 pmol protein 33.
Step 8: Post-translational modification Glycosylation is a common posttranslational modification and may be quite extensive in certain proteins. Glycosylation sites are usually detected by the inabi]ity of Edman degradation to produce a detectable PTH derivative of glycosylated amino acids. Endoglycosidase digestion 34 is needed to identify the PTH derivative of a deglycosylated amino acid. Although elucidation of the structure of the released saccharide chain would be desirable, a routine method for microanalysis at levels similar to those of amino acid sequencing has not been developed. Nevertheless, it may be possible to deduce the structures of some oligosaccharides released from glycoproteins based on the retention times
By these methods a protein/peptide is ionized (positively or negatively or both) without fragmentation, making it possible to detect a molecular ion.
Fast atom bombardment (FAn): Samples are placed in a matrix (glycerol) for low energy ionization with a beam of neutral atoms (Ar or Xe).
Plasma desorption (PD): Solid samples are placed on a metal foil or a nitrocellulose membrane (for protein/peptide) for high energy ionization (MeV levels) with a beam of fission fragments of 252Cf. Liquid secondary ion: Samples in liquid matrix irradiated by positively charged Ar, Xe or Cs form secondary molecular ions; energy for ionization at keV levels. Laser desorption: Sample (solid or in liquid matrix)irradiated with laser beam to ionize molecules of high masses with low stability. Electrospray: Useful for large proteins, produces multiple charged molecular i o n s 24.
Tandem mass spectrometry: Two mass spectrometers, tandemly connected, used to determine the covalent structure of a molecule. The first instrument is a sepa rater for fragments of the proteolytic digest of a protein with respect to their masses. A fragment thus separated is introduced to a chamber for fragmentation (collision-induced degradation) and the masses of subfragments formed are measured with the second spectrometer. The 'collisioninduced degradation' takes places when charged molecules collide with neutral atoms (such as He). In the case of a peptide, the analysis of the masses of subfragments leads to the determination of its amino acid sequence.
on HPLC columns of their fluorescent 2-aminopyridyl derivatives 35,36. Mass spectrometry has also been used for the structural analysis of such saccharides in addition to other typical side chain modifications such as phosphorylation and methylation, acetylation. The structure of oligosaccharide p-ethoxycarbonylphenylglycosides has been determined by mass spectrometry 37 using only 5 pmol of sample. Protein sequencing and mass spectrometry Soft ionization mass spectrometry (see Box) is a powerful technique which can complement the sequencing methodology based on Edman degradation, by facilitating the assessment of post-translational modifications and the speed of analysis. However, FAB mass spectrometry, with which protein chemists are most familiar, is relatively insensitive and may require 0.1-0.5 nmol of the sample for a single measurement. Tandem mass spectrometry (see Box) requires 0.32 nmol to determine one amino acid residue. At present, the sensitivity of mass spectrometry is much lower than that of Edman degradation 9. Nevertheless, tandem mass spectrometry is used to compare the structures of closely related N ~blocked proteins 38 and to confirm the primary structure of a recom-
binant protein 39. The upper limit of molecular mass analysed routinely by mass spectrometry is 5 kDa and 15 20 kDa for the FAB and 252CfPD method 4°, respectively. The measurable mass and the sensitivity are rapidly increasing; ultraviolet-laser ionization/desorption mass spectrometry could detect the molecular peak of a 175 kDa protein as little as 50 fmol (Ref. 41). Electrospray can also determine precisely the molecular mass of large proteins 24. It is important for protein chemists to decide which soft ionization method is most suited for his or her structural analysis. Disadvantage of mass spectrometry is the lack of information on the quantity of a protein/ peptide detected as a peak. This is substantially offset by chemical analysis of the composition of a protein, the importance of its precise amino acid analysis being stressed in protein mass spectrometry. Prediction of protein function based on sequence homology search Searching for sequence homology in protein sequence databases is now a routine procedure. Such searches have revealed sequence similarities between many apparently unrelated proteins, or proteins whose function is unknown. Recently, we predicted that a glycoprotein associated with gametophytic self-incompatibility in pollination of Nicotiana alata is a
TIBTECH - O C T O B E R 1990 [Vol. 8]
ribonuclease, based solely on the result of a search for sequence homology w i t h ribonuclease T242: Two short sequences were identical bet w e e n these two proteins and each of t h e m i n v o l v e d one of the two essential histidine residues in ribon u c l e a s e T2. The p r e d i c t i o n was s u b s e q u e n t l y confirmed by d e m o n strating the ribonucleolytic activity of the g l y c o p r o t e i n 43. Thus, in addition to the d e t e r m i n a t i o n of amino acid s e q u e n c e and the analysis of s e q u e n c e information, protein seq u e n c e analysis in the near future s h o u l d integrate the test of the function p r e d i c t e d for a protein in question.
Conclusion With the present status of protein s e q u e n c i n g involving E d m a n degradation, the complete a m i n o acid s e q u e n c e of a 10 kDa protein can be d e t e r m i n e d by a skilled protein chemist, w h e n 1 nmol of a highly p u r e sample is available in a welle q u i p p e d laboratory. However, elucidation of the entire covalent structure i n c l u d i n g side chain modification needs a m u c h larger a m o u n t of sample because of the lack of established microanalysis technologies r o u t i n e l y applicable for all n e c e s s a r y structural analysis. A variety of soft ionization mass s p e c t r o m e t r y is surely taking part in this analysis. It is possible to use n e w tools described here for partial s e q u e n c e analysis of a protein blotted to a m e m b r a n e , w h i c h will serve for the rapid, extensive structura| analysis of proteins purified by SDS-PAGE. T h e final goal of protein sequence analysis is to establish an a u t o m a t e d system for the rapid, precise analysis of the covalent structure of a protein w i t h the least c o n s u m p t i o n of the sample. E d m a n degradation and mass s p e c t r o m e t r y will be major m e a n s for protein sequencing in the next decade, their rapid reformation to the ultramicroanalysis m e t h o d s being strongly investigated. For seq u e n c i n g by either method, protein fragmeritation to peptides of appropriate size is essential. A search for and the synthesis of n e w proteases c o m p a r a b l e in specificity with restriction e n d o n u c l e a s e s specific to each of the 20 amino acids is important for protein sequencing and protein engineering in the future.
287
Note a d d e d in p r o o f T h r e e interesting articles and s y m p o s i u m proceedings have recently b e e n published. These deal w i t h electrospray ionization 44 and its c o m b i n a t i o n with ion trap mass s p e c t r o m e t r y 45, and ion trap mass spectrometry alone 46, looking t o w a r d p r o t e i n m i c r o s e q u e n c i n g at s u b p i c o m o l a r concentrations. Advances in sequencing t e c h n i q u e s have also r e c e n t l y been covered in Ref. 47.
References 1 Hugli, T. E. (ed.) (1989) Techniques in Protein Chemistry, Academic Press 2 Matsudaira, P. T. (ed.) (1989) A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press 3 Wittmann-Liebold, B. (ed.) (1989) Methods in Protein Sequence Analysis Springer-Verlag 4 Findley, J. B. C. and Geisow, M. J. (eds) (1989) Protein Sequencing: A Practical Approach, IRL Press 5 Allen, G. (1989) Sequencing of Proteins and Peptides, 2nd edn, Elsevier 6 Shively, J. E., Paxton, R. J. and Lee, J. D. (1989) Trends Biochem. Sci. 14, 246-255 7 Yuen, S. W., Chui, A. H., Wilson, K. J. and Yuan, P. M. (1989) Biotechniques 7, 74-82 8 Tsunasawa, S., Takakura, H. and Sakiyama, F. (1990) J. Protein Chem. 9, 265-266 9 Biemann, K. (1988) Biomed. Environ. Mass Spectrom. 16, 99-111 10 Ganno, S., Wakabayashi, K., Yagi, Y. and Sakiyama, F. (1990) in High Performance Liquid Chromatography of Proteins and Peptides II (Ikenaka, T. and Sakiyama, F., eds), pp. 3-10, Kagaku Dojin (in Japanese) 11 Tous, G. I., Fausnaugh, J. L., Akinyosoye, O., Lackland, H., WinterCash, P., Victorica, F. J. and Stein, S. (1989) Anal. Biochem. 179, 50-55 12 Yu, M. and Dovichi, N. J. (1989) Anal. Chem. 61, 37-40 13 Kennedy, R. T., Oates, M. D., Cooper, B. R., Nickerson, B. and Jorgenson, J. W. (1989) Science 246, 57-63 14 Liu, T-Y. and Boykins, R. A. (1989) Anal. Biochem. 182, 383-387 15 Masaki, T., Fujihashi, T., Nakamura, K. and Soejima, M. (1981) Biochim. Biophys. Acta 660, 51-55 16 Kawata, Y., Sakiyama, F. and Tamaoki, H. (1988) Eur. J. Biochem. 176, 683-697 17 Tsunasawa, S., Masaki, T., Hirose, M., Soejima, M. and Sakiyama, F. (1989) J. Biol. Chem. 264, 3832-3839 18 Maier, G., Drapeau, G. R., Doenges,
19 20
21 22
23 24 25 26 27 28 29 30 31
32 33
34 35 36 37
38 39
K-H. and Ponstingl, H. (1987) in Methods in Protein Sequence Analysis 1986 (Walsh, K. A., ed.), pp. 335-337, Humana Press Chen, L., Cotter, R. J. and Stults, J. T. (1989) Anal. Biochem. 183, 190-194 Hafok-Peters, Ch., Maurer-Fogy, I. and Schmid, E. R. (1990) Biomed. Environ. Mass Spectrom. 19, 159-163 Caprioli, R. M., Moore, W. T., Dague, B. and Martin, M. (1988) J. Chromatogr. 443, 355-362 Grossmann, P. D., Colburn, J. C., Lauer, H. H., Nielsen, R. G., Riggin, R. M., Sittampalam, G. S. and Rikard, E. C. (1989) Anal. Chem. 61, 1186-1194 Cobb, K. A. and Novotny, M. (1989) Anal. Chem. 61, 2226-2231 Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. and Whitehouse, C. M. (1989) Science 246, 64-71 Aoyama, H., Iwamatsu, A., Dib6, G., Tsunasawa, S. and Sakiyama, F. (1988) J. Protein Chem. 7, 191 Tsugita, A., Arai, I., Kamo, M. and Jone, C. S. (1988) J. Biochem. 103, 399-401 Yamamoto, A., Toda, H. and Sakiyama, F. (1989) J. Biochem. 106, 552-554 Rose, K., Savoy, L. A., Simona, M. G., Offord, R. E. and Wingfield, P. (1988) Biochem. J. 250, 253-259 Knmazaki, T., Fujita, A., Terasawa, K., Shimura, K., Kasai, K. and Ishii, S. (1987) J. Biochem. 102, 1539-1546 Ohuchi, C., Kondo, J. and Hishida, T. (1988) Seikagaku 60, 875 (in Japanese) Miller, C. G., Kong, C. and Shively, J. E. (1989) in Techniques in Protein Chemistry (Hugli, T. E., ed.), pp. 67-78, Academic Press Sun; Y. and Smith, D. L. (1988) Anal. Biochem. 172, 130-138 Rodriguez, H., Nevins, B. and Chakel, J. (1989) in Techniques in Protein Chemistry (Hugli, T. E., ed.), pp. 186-194, Academic Press Maley, F., Trimble, R. B., Tarentino, A. L. and Plummer, T. H. (1989) Anal. Biochem. 180, 195-204 Tomiya, N, Awaya, J., Kurono, M., Endo, S., Arata, Y. and Takahashi, N. (1988) Anal. Biochem. 171, 73-90 Oku, H., Hase, S. and Ikenaka, T. (1990) Anal. Biochem. 185, 331-334 Poulter, L., Earnest, J. P., Stroud, R. M. and Burlingame, A. L. (1988) Biomed. Environ. Mass Spectrom. 16, 25-30 Hunt, D. F., Yates III, J. R., Shabanov, J., Bruns, M. E. and Bruns, D. E. (1989) J. Biol. Chem. 264, 6580-6536 Furuya, M., Akashi, S. and Hirayama, K. (1989) Biochem. Biophys. Res. Commun. 163, 1100-1106
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40 Roepstorff, P. (1989) Acc. Chem. Res. 22,421-427 41 Karas, M., lngendoh, A., Bahr, U. and Hillenkamp, H. (1989) Biomed. Environ. Mass Spectrom. 18,841-843 42 Kawata, Y., Sakiyama, F., Hayashi, F. and Kyogoku, Y. (1990) Eur. J. Biochem. 187, 255-262 []
[]
[]
43 Mclure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F. and Clarke, A. E. (1989) Nature 342,955-957 44 Smith, R. D., Loo, J. A., Edmonds, C. G., Barinaga, C. J. and Udseth, H. R. (1990) Anal. Chem. 63,882-889 45 Van Berkel, G. J., Glish, G. L. and []
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Biochemical reactions in supercritical fluids
McLucky, S. A. (1990) Anal. Chem. 62, 1284-1295 46 Cooks, R. G. and Kaiser, R. E., Jr (1990) Acc. Chem. Res. 23, 213-219 47 Villafranca, J. J. (ed.) (1990) Current Research in Protein Chemistry: Techniques, Structure and Function, Academic Press []
[]
[]
--Fig. 1 250
I
I
75°1C~ " j
20(
Kozo Nakamura
150
Supercritical fluids (SCF), which have been gaining recognition as suitable solvents for extraction of biomolecules, also show promise for use as novel media for enzymatic reactions. Their advantages, compared with organic solvents, include, among others, easily controllable solubility of components, high diffusivity, low toxicity and improved reaction rates. The use of SCF should facilitate the integration of biochemical reactions and separation processes.
0
100
200
300
400
500
Pressure (MPa)
All materials have critical points which determine their phase behaviour: the critical temperature (To) is that above which a gas cannot be liquefied by increasing pressure; the critical pressure (Pc) is that at which, at the critical temperature, a liquid may exist in equilibrium with the gas phase. For example, Tc and Pc for carbon dioxide are 31.0°C and 73.8 bar, respectively. Supercritical fluids (SCF) are those which exist as fluids at temperatures and pressures above their critical points. They are of interest in industrial processing since their solvent properties vary considerably with relatively minor changes in temperature and pressure. A recent review by Randolph 1 indicated that the advantages of SCF solvents for extraction could outweigh such disadvantages as lower solubilities and higher capital costs compared with liquid solvents. Many of the properties of SCF which suit them for use in extraction processes also render them attractive as a medium for biocatalytic reactions:
K. Nakamura is at the Department of Biological and Chemical Engineering, Gunma University, Kiryu, Gunma 376, Japan.
• high diffusivity and low surface tension lead to reduced internal mass-transfer limitations for heterogeneous chemical or biochemical catalysts; • the dramatic sensitivity of solubility to changes in pressure and temperature may be used to manipulate the selectivity of a reaction and to recover a product from reactants. The solubility or the state of solute molecules may also be changed with the addition of a small amount of properly chosen co-solvent to control the reaction kinetics; • The pressures associated with suitable SCF such as supercritical CO2 (SCCO2) or ethane are not so high as to damage biopolymers, while the temperatures required are appropriately low for thermally labile biomolecules; • the use of SCF is not accompanied by the problem of solvent residues in the reaction product because SCF solvents are gases under atmospheric conditions. In particular, the low toxicity and reactivity of SCCO2 make it attractive as a non-aqueous reaction medium for food and pharmaceutical products. SCF as media for chemical reactions have been reviewed by
@ 1990, Etsevier Science Publishers Ltd (UK) 0167 - 9430/90/$2.00
Solubility of water in high pressure carbon dioxide: comparison of values derived using Eqn 1 (lines), with experimental data (symbols) obtained by Wiebe et al. 4
--Fig. 2 f6ooc 3O 25
-~
20
~ 15 40°C 10
5 o
25°C F
o
20
I
4O Pressure
I
60 (MPa)
I
80
100
Solubility of stearic acid in high pressure carbon dioxide, calculated using Eqn 1.
Subramaniam and McHugh 2, with examples of hydrolysis, thermal and photochemical reactions, isomerization and heterogeneous catalytic
T I B T E C H - O C T O B E R 1990 [Vol. 8]
Reply from Blalock I entirely agree with J. Slootstra and E. Roubos that 'receptor and hormone may have originated from a single DNA molecule in which one strand coded for the hormone and the other for the receptor'. In fact, our original article stated this 1. Similarly, we concur 2 with R. Brentani that co-evolution of interacting peptides coded for by comp-
lementary codons would speed up evolution. I do, however, disagree with his contention that the X-ray crystallography and site-specific mutagenesis work was misquoted. These techniques led to identification of the same sites of interaction of interleukin-2 and its receptor as would be predicted by the Molecular Recognition Theory.
New tools for protein sequence analysis Fumio Sakiyama Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation w h i c h facilitate determination of protein primary structure have recently been developed. The functional role of a protein depends on its specific threedimensional structure which is determined primarily by its amino acid sequence (primary structure). Protein sequence analysis is thus an essential step towards an understanding of a protein's function. Since the amino acid sequence is dictated by the nucleotide sequence, DNA sequencing (now a rapid, straightforward technique, relatively economical in lerms of materials required) has become a popular method of obtaining information on amino acid sequence, especially for large proteins of which only small amounts are a v a i l a b l e for study. However, this approach has the drawback that it provides no information on the structure of proteins modified post-translationally. Direct sequencing of the polypeptide chain(s) is therefore a preF. Sakiyama is at the Institute for Protein Research, Osaka University, Saita, Osaka 565, Japan.
ferred option when only nanomolar amounts of highly purified protein of molecular mass of 30 kDa or less are available. Recently, new instrumentation and improved techniques have been developed I 6. We discuss those which are now being used routinely as well as those which will become of practical importance in the near future. The strategy for protein sequence analysis has remained essentially unchanged for the past 30 years, and employs a series of reactions termed the Edman degradation. I n this, phenylisothiocyanate is reacted with the terminal amino group of a protein/peptide yielding a derivative which may be cleaved from the peptide chain. The cleaved residue undergoes a rearrangement to a phenylthiohydantoin (PTH) derivative of the amino acid, easily identified by HPLC. Repetition of this sequence of reactions permits the sequencing of the complete polypeptide, starting from the N-terminal
1990, Elsevier Science Publishers Ltd (UK) 0167- 9430/90/$2.00
References 1 Bost, K. L., Smith, E. M. and Blalock, J. E. (1985) Proc. Natl Acad. Sci. USA 82, 1372-1375 2 Blalock, J. E. and Bost, K. L. (1988) Recent Prog. Horm. Res. 44, 199-222 J. E D W I N B L A L O C K
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
end. Nowadays the majority of direct sequencing by Edman degradation is carried out in sequenators with most of the process automated.
Protein sequence analysis procedure A typical protein sequence analysis procedure consists of eight steps: (1) N-terminal analysis; (2) amino acid analysis; (3) protein fragmentation; (4) peptide fractionation; (5) Edman degradation and PTH-amino acid analysis; (6) C-terminal analysis; (7) location of the disulfide bond; and (8) determination of the mode and site of post translational modification. Step 1: N-terminal analysis This can be u s e d not only to determine the amino acid sequence of the N-terminal region but also to test the purity of the protein sample. A gas-phase or pulsed-liquid sequencer (see Glossary~ equipped with PTH-amino acid analyser can determine the 30-40 N-terminal amino acids by direct sequencing of 50-100 pmol of a protein. Under optimal running conditions, up to 10 amino acid residues may be determined from 5-10 pmol of a protein. The N-terminal sequencing of a protein which has been separated by SDS-PAGE and blotted to a polyvinylidenedifluoride (PVDF) (see Glossary) membrane is routine 7. Frequently, in the direct sequence analysis, a PTH-amino acid cannot be detected in the first cycle of Edman degradation of a protein. In most cases, this is due to the reaction being blocked by an acyl group at the N-terminus. The presence of an acetyl group at the N-terminus is a
TIBTECH- OCTOBER 1990 [Vol. 8]
283
~Fig. mGIossary Polyvinylidenedifluoride (PVDF) m e m b r a n e - teflon-like m a t e r i a l n o t d a m a g e d by reagents and s o l v e n t s used in c o m m e r c i a l automated sequencing instruments. Proteins bind to the m e m b r a n e t h r o u g h h y d r o p h o b i c interactions and can be visualized by staining w i t h dyes. C o n t a m i n a n t s can be w a s h e d f r o m the m e m b r a n e by rinsing w i t h w a t e r . P u l s e d liquid (or pulse liquid) protein s e q u e n c e r - indicates that a m i n u t e (but quantified) a m o u n t of a reagent in liquid is s u p p l i e d like a pulse to the m e d i u m of E d m a n reaction. Before the i n t r o d u c t i o n of this m e t h o d the reagent w a s s u p p l i e d , u n q u a n t i t a t i v e l y , as a gas (gas-phase protein sequencer).
1 E, R2 i I ECO-NH-CH-CO-NH-CtI-CO- - -
R2
I I RCO-NH-CII-CO-N!t-CH-CO- - -
-Lys + NH~ - - -Lys + Nil- -
1
Phenylisothiocyanate
R, E2 I I RCO-NH-C!t-CO-NH-CH-CO. . . .
PTC PTC I I Lys 4 PTC-NH- - - L y s + PTC-NH- -
Performic acid oxidation
R, R2 I 1 RCO-Nt-gtt-CO-NH-CIf-go . . . .
PC I l,ys +
PC I PC-Nit- - - g y s + PC-NH- -
A A R E digestion
RCO-NtI-CH-COOit
Step 2: Amino acid analysis Amino acid analysis is essential to determine the composition of the intact protein and its peptide fragments. Precision has priority over sensitivity in routine amino acid analysis and the established ninhydrin method is well-suited for precise analysis, but the amount of protein (/> 100 pmol) necessary for quantitative analysis by this method
Lys-
API digestion
RI
E,
common modification of intracellular proteins. The acetyl group can be removed with acylamino acid-releasing enzyme (AARE) as acetylamino acid. However, AARE attacks only N~-acylated peptides of less than 40 residues and the N-acety]ated protein must be fragmented into peptides prior to enzyme digestion. Recently, we devised a procedure for removing the N-terminal acetylamino acid without isolation of the acylated peptide 8 (see Fig. 1). The same principle may be applied for removal of the N-terminal pyroglutamyl group, using pyroglutamylaminopeptidase instead of AARE. Confirmation of the chemically determined sequence of the N~-acylated peptide by mass spectrometry using soft ionization (see Box), mainly fast atom bombardment (FAB) and plasma desorption (PD), has become routine 9.
-I,ys. . . . .
Identification of N-acetylamino acid
+
122 I NH~-CH-CO. . . .
Pg I Lys
+
PC I PC-NH- - -l,ys + PC-Nil- -
A m i n o acid s e q u e n c e analysis
The N-terminal sequence analysis of an N
is large compared with the amounts required for protein sequence analysis. Optimization of the ninhydrin method* has enabled us to determine the amino acid composition (except for Pro) of a protein within + 5% for 50 pmol levels (Fig. 2) l°. Several post- and pre-column derivatization methods (Table 1) are used for high-sensitivity analysis at picomolar methods where error often increases beyond 10%. At least 10 pmol of the protein/peptide sample (including samples obtained by on-the-membrane vapor-phase hydrolysis; i.e. hydrolysis of a pro-
*The sensitivity of the amino acid analysis was optimized by modifying two important reaction steps. One was to increase the signal-to-noise ratio by optimizing the amino acid-ninhydrin colorization reaction and the other was to use gradient elution of amino acids instead of step elution to ensure baseline separation of each amino acid.
tein/peptide blotted on a membrane [such as PVDF] with gaseous hydrochloric acid) 11 is needed to give reproducible and reasonably accurate determinations in most of the high-sensitivity methods though their limit of detection is less than a tenth of the values shown in Table 1. The detection limit of amino acids may now be as low as the femtomole or attomole level for some methods; for example analysis using a set of capillary zone electrophoresis (CZE) and 4-dimethyl-aminoazobenzene-4'sulfonyl amino acids 12 or a set of CZE or open tubular liquid chromatography (OTLC) and laser-induced fluorescence detection of isoindole compounds that had been formed upon reaction with naphthalene-l,2dialdehyde of amino acids 13. In OTLC, the separation takes place inside a capillary tube (inner diameter of 1-50 gm; length I> 1 m), the stationary phase being attached
284
TIBTECH- OCTOBER 1990 [Vol. 8]
--Fig. 2 440nm
to the inner wall of the tube (instead of packed particles in the case of the conventional HPLC). OTLC has an advantage over CZE in that the separated material can be collected and used in subsequent experiments. However, in high sensitivity quantitative analysis using picomolar (or lower) amounts, a serious problem exists: how can one collect reliable data for the 'clean' sample, free from contamination by glycine, serine and their derivatives in the course of the purification and hydrolysis of a protein/peptide. Even deionized, distilled water is a possible source of contamination at these levels. In addition to the vapor-phase acid hydrolysis, the liquidphase hydrolysis of protein/peptide in a minimal amount of hydrochloric acid in sealed capilary tubes 14 may help to decrease this unfavorable amino acid contamination.
Step 3: Fragmentation Two proteases Achromobacter protease I (API or lysylendopeptidase) and Asp-N, each of which recognizes specific amino acid residues, have recently been introduced and used successfully in the fragmentation of proteins and peptides. API is a serine protease hydrolysing lysyl peptide bonds, including the Lys-Pro bond 15 and the S-aminoethylcysteinyl bond 16. Characteristics such as its proteolytic activity (greater than trypsin), stability in the presence of 5 M urea and 0.1% SDS, and wide pH optimum (pH 8.5-10.7) make it favored as an enzyme for protein fragmentation. API is also active at pH 6.5 where thio[-disulfide exchange is suppressed. API (consisting of 268 residues) 17, is more active and more stable than the endoproteinase Lys-C and is routinely used for the primary fragmentation of a protein in our laboratory. To prepare sufficient peptide fragments to cover the entire peptide chain, a combination of API and Staphylococcus aureus V8 protease digestion is used. Asp-N, a bacterial metalloprotease, hydrolyses the peptide bond at the N-terminal side of aspartic acid and cysteic acid 18 and is suited for subfragmentation of large peptides. Proteases and chemical reagents of practical importance in protein/ peptide fragmentation are listed in Table 2.
lOOpmol
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1
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r
I
~
i
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I
I
I
10
15
20
25
30
35
40
45
50
55
Time (min)
Chromatograms of 16 protein amino acids recorded with a Hitachi L8500S amino acid analyser by the ninhydrin method (50 and 100 pmol sample for each amino acid). Ordinate is absorbance at 570 nm. Inset on the upper chromatogram is for proline (200 pmol, 12.61 min) detected at 440 nm.
--Table I Typical methods for amino acid analysis by pre- and post-column derivatization A m i n o acid
derivatives or
Derivatization Detection A p p r o x i m a t e l o w e r (nm) limit of quantitation
reagents Ninhydrin PhenylthiocarbamoylA A b (PTC) 4-Dimethylaminoazobenzene-4'-sulfonylA A (Dabsyl) o-Phthaldehyde + RSH (OPA) (fluorescent) 9-FluorenylmethyloxycarbonyI-AA (Fmoc) (fluorescent)
(pmol) a post pre
570 254
50 10
pre
436
10
pre post pre
ex. 340 em.455 ex. 264 em.340
aValues are for routine analysis. bAbbreviations: AA, amino acids; ex., excitation; em., emission.
5-10 10
TIBTECH- OCTOBER1990[Vol.8]
285
- - T a b l e 2, Methods for the cleavage o f the peptide chain Proteases
Scissile bonds
Proteases/ reagents
Scissile bonds
API Lys-X Subtilisin nonspecific (Lysylendopeptidase)a Endoproteinase Lys-C Papain Lys-X nonspecific Trypsin Pepsin Arg/Lys-X nonspecific Arginylendopeptidase Arg-X Clostripain Arg-X BrCN Met-X V8 proteaseb Glu-X H+/H20 Asp-Pro Asp-N X-Asp Iodosobenzoic acid Trp-X Prolylendopeptidase Pro-X Asn-Gly NH2OH Thermolysin X-Leu/Val/lle/Met NTCBc or DMAPX-Cys CNd Chymotrypsin Tyr/Tr p/Phe/Leu-X X-Asp/Asn-Y H+/H20,heat aAvailablefrom Wako PureChemical Industries bStaphylococcusaureus C2-nitro-5-cyanothiobenzoicacid 1-cyano-4-dimthylaminopyridiniumperchlorate
Step 4: Peptide fractionation Before reverse-phase HPLC was introduced, peptide purification was a time-consuming and complicated part of protein sequencing. Although the use of HPLC saves time and effort in peptide purification, the recovery of peptides from a reverse phase column is in the range of only 3080%. In particular, hydrophobic peptides are often not eluted (or only with difficulty) from the column. To detect a possible missing peptide(s), FAB mass spectrometry is useful since hydrophobic peptides of 5 kDa and less are more easily dispersed than hydrophilic ones. Conversely, hydrophilic peptides adsorbed on a nitrocellulose membrane tend to desorb more easily than hydrophobic ones by 252Cf PD 19. Proteolysis of a protein on the probe tip of a FAB mass spectrometer saves time and sample 2o. In any event, a comparison of two 'mass summaries' obtained before and after reverse phase HPLC of the proteolytic digest of a protein is recommended to detect possible missing peptides 21. Another technique complimentary to reverse phase HPLC for peptide mapping is CZE 22. As little as 50 ng of protein is digested with trypsin, immobilized in a packed column and mapped by CZE 2a. Attempts have been made to construct the online mass spectral analysis system of a peptide mixture. When the formation of large fragments is anticipated in protein fragmentation, a combination of electrospray ionization mass spectrometry 24 and CZE or OTLC seems promising, since, in electrospray ionization, a protein
taining SDS at pH 3.11. The limit of detection of PTH-amino acids is as little as 500 fmol with a conventional HPLC apparatus. A more sensitive method has also been reported for the determination of the N-terminal amino acid released by Edman degradation. This method utilizes the ring-opening reaction of anilinothiazolinone by 4-aminofluorescein to a fluorescent phenylthiocarbamoylamino acid amide, which is detected at the attomolar level 26.
Step 6: C-terminal analysis
Few good methods for C-terminal analysis exist; hydrazinolysis and carboxypeptidase Y digestion are the only practicable means. Hydrazinolysis, which results in cleavage and sprayed from the end of a capillary derivatization to amino acid hydracolumn to a high electric field zine (with all except the C-terminal (3-4 kV) becomes a group of mul- residue being unmodified) has retiple charged molecules which can cently been improved as vaporbe detected in the limited range of phase hydrazinolysis. As a result, small mass-to-charge ratio (m/z). the duration of reaction has been Accordingly, this separation- shortened to 2-4 h, the detection detection method detects all pep- sensitivity improved and the recovery tide fragments including large pep- of labile amino acids such as Ser, tides which might be missed by FAB Thr and Asp increased 27. Carboxymass spectrometry. peptidase Y, unlike other carboxypeptidases which show some speciStep 5: Edman degradation and ficity in cleavage, will cleave any PTH-amino acid analysis C-terminal residue and may be used Protein sequencing is usually per- to identify the first few C terminal formed using a gas-phase or pulsed- residues sequentially. liquid sequencer with an on-line Three n e w methods for C-terminal PTH-amino acid analysis system. analysis have been reported. The Unlike solid-phase sequenators first one uses acid hydrolysis in where the polypeptide is coupled H21ao; only the C-terminal amino through its C-terminus to a support- acid remains isotopically unlabeled, ing matrix, in gas-phase sequencing and is easily distinguished by mass the polypeptide is strongly adsorbed spectrometry from the internal and on a glass fiber or PVDF paper, and N-terminal amino acids of a prothe Edman chemistry is performed tein 28. The two other methods inusing trimethylamine and trifluoro- volve the isolation of the C-terminal acetic acid in vapor phase, permit- peptide by means of specific adsorpting easier handling and reduced tion of the other peptides to an contamination. Although auto- appropriate solid support. One supmation of the Edman reaction for port is a dehydrotrypsin-liganded sequencing is well established, diffi- column which has no affinity for the culties such as the wash-out of part C-terminal peptide lacking Arg and of hydrophobic peptides, the partial Lys in the tryptic digest of a prorandom hydrolysis of peptide bond tein 29. (When the C-terminus of a with trifluoroacetic acid and the o~,[3- protein is Arg or Lys, the affinity rearrangement of the Asn-Gly pep- column also adsorbs the C-terminal tide bond still remain. Since the peptide and this method cannot be number of amino acids determined used directly for C-terminal analysis.) and the reliability of the result of The other support is p-phenylenesequencing depend mainly on the diisothiocyanate (DITC)-activated sensitivity and the reproducibility of glass, to which peptides in the APIPTH-amino acid analysis, we have digest of a protein are first bound devised 25 an isocratic elution analy- covalently and then the C-terminal sis recycling a formate buffer con- peptide is selectively released by
286
TIBTECH - OCTOBER 1990 [Vol. 8]
--Box I Soft ionization mass spectrometry techniques
one cycle of Edman degradation 3°. This works well when Lys is absent from the C-terminus. Efforts have also been made to improve the thiohydantoin method, which releases the C-terminus of a protein as amino acid thiohydantoin via the formation of a mixed anhydride of the C-terminal carboxyl group with isothiocyanic acid. At present, this is a sole potential candidate for the C-terminal Edmantype sequencing. However, the method has not grown to become routine for microanalysis at picomolar amounts 31 mainly because of its low efficiency of performance relative to Edman degradation in terms of sensitivity and the number of determinable amino acid residues.
Step 7: Location of disulfide bonds The determination of disulfide bonds in a protein using chemical methods is time-consuming and often requires nanomolar amounts of sample. Mass spectrometry is the prefered method for the rapid determination of disulfide bonds in a peptide whose amino acid sequence is known; the technique is based on the detection of a peak(s) whose m/z value changes before and after the reduction with 1,4-dithiothreitol or the performic acid oxidation of disulfide bonds 32. The on-the-probe cleavage of disulfide bonds enables the analysis to be very sensitive, and the location of disulfide bonds can be determined using just 100 pmol protein 33.
Step 8: Post-translational modification Glycosylation is a common posttranslational modification and may be quite extensive in certain proteins. Glycosylation sites are usually detected by the inabi]ity of Edman degradation to produce a detectable PTH derivative of glycosylated amino acids. Endoglycosidase digestion 34 is needed to identify the PTH derivative of a deglycosylated amino acid. Although elucidation of the structure of the released saccharide chain would be desirable, a routine method for microanalysis at levels similar to those of amino acid sequencing has not been developed. Nevertheless, it may be possible to deduce the structures of some oligosaccharides released from glycoproteins based on the retention times
By these methods a protein/peptide is ionized (positively or negatively or both) without fragmentation, making it possible to detect a molecular ion.
Fast atom bombardment (FAn): Samples are placed in a matrix (glycerol) for low energy ionization with a beam of neutral atoms (Ar or Xe).
Plasma desorption (PD): Solid samples are placed on a metal foil or a nitrocellulose membrane (for protein/peptide) for high energy ionization (MeV levels) with a beam of fission fragments of 252Cf. Liquid secondary ion: Samples in liquid matrix irradiated by positively charged Ar, Xe or Cs form secondary molecular ions; energy for ionization at keV levels. Laser desorption: Sample (solid or in liquid matrix)irradiated with laser beam to ionize molecules of high masses with low stability. Electrospray: Useful for large proteins, produces multiple charged molecular i o n s 24.
Tandem mass spectrometry: Two mass spectrometers, tandemly connected, used to determine the covalent structure of a molecule. The first instrument is a sepa rater for fragments of the proteolytic digest of a protein with respect to their masses. A fragment thus separated is introduced to a chamber for fragmentation (collision-induced degradation) and the masses of subfragments formed are measured with the second spectrometer. The 'collisioninduced degradation' takes places when charged molecules collide with neutral atoms (such as He). In the case of a peptide, the analysis of the masses of subfragments leads to the determination of its amino acid sequence.
on HPLC columns of their fluorescent 2-aminopyridyl derivatives 35,36. Mass spectrometry has also been used for the structural analysis of such saccharides in addition to other typical side chain modifications such as phosphorylation and methylation, acetylation. The structure of oligosaccharide p-ethoxycarbonylphenylglycosides has been determined by mass spectrometry 37 using only 5 pmol of sample. Protein sequencing and mass spectrometry Soft ionization mass spectrometry (see Box) is a powerful technique which can complement the sequencing methodology based on Edman degradation, by facilitating the assessment of post-translational modifications and the speed of analysis. However, FAB mass spectrometry, with which protein chemists are most familiar, is relatively insensitive and may require 0.1-0.5 nmol of the sample for a single measurement. Tandem mass spectrometry (see Box) requires 0.32 nmol to determine one amino acid residue. At present, the sensitivity of mass spectrometry is much lower than that of Edman degradation 9. Nevertheless, tandem mass spectrometry is used to compare the structures of closely related N ~blocked proteins 38 and to confirm the primary structure of a recom-
binant protein 39. The upper limit of molecular mass analysed routinely by mass spectrometry is 5 kDa and 15 20 kDa for the FAB and 252CfPD method 4°, respectively. The measurable mass and the sensitivity are rapidly increasing; ultraviolet-laser ionization/desorption mass spectrometry could detect the molecular peak of a 175 kDa protein as little as 50 fmol (Ref. 41). Electrospray can also determine precisely the molecular mass of large proteins 24. It is important for protein chemists to decide which soft ionization method is most suited for his or her structural analysis. Disadvantage of mass spectrometry is the lack of information on the quantity of a protein/ peptide detected as a peak. This is substantially offset by chemical analysis of the composition of a protein, the importance of its precise amino acid analysis being stressed in protein mass spectrometry. Prediction of protein function based on sequence homology search Searching for sequence homology in protein sequence databases is now a routine procedure. Such searches have revealed sequence similarities between many apparently unrelated proteins, or proteins whose function is unknown. Recently, we predicted that a glycoprotein associated with gametophytic self-incompatibility in pollination of Nicotiana alata is a
TIBTECH - O C T O B E R 1990 [Vol. 8]
ribonuclease, based solely on the result of a search for sequence homology w i t h ribonuclease T242: Two short sequences were identical bet w e e n these two proteins and each of t h e m i n v o l v e d one of the two essential histidine residues in ribon u c l e a s e T2. The p r e d i c t i o n was s u b s e q u e n t l y confirmed by d e m o n strating the ribonucleolytic activity of the g l y c o p r o t e i n 43. Thus, in addition to the d e t e r m i n a t i o n of amino acid s e q u e n c e and the analysis of s e q u e n c e information, protein seq u e n c e analysis in the near future s h o u l d integrate the test of the function p r e d i c t e d for a protein in question.
Conclusion With the present status of protein s e q u e n c i n g involving E d m a n degradation, the complete a m i n o acid s e q u e n c e of a 10 kDa protein can be d e t e r m i n e d by a skilled protein chemist, w h e n 1 nmol of a highly p u r e sample is available in a welle q u i p p e d laboratory. However, elucidation of the entire covalent structure i n c l u d i n g side chain modification needs a m u c h larger a m o u n t of sample because of the lack of established microanalysis technologies r o u t i n e l y applicable for all n e c e s s a r y structural analysis. A variety of soft ionization mass s p e c t r o m e t r y is surely taking part in this analysis. It is possible to use n e w tools described here for partial s e q u e n c e analysis of a protein blotted to a m e m b r a n e , w h i c h will serve for the rapid, extensive structura| analysis of proteins purified by SDS-PAGE. T h e final goal of protein sequence analysis is to establish an a u t o m a t e d system for the rapid, precise analysis of the covalent structure of a protein w i t h the least c o n s u m p t i o n of the sample. E d m a n degradation and mass s p e c t r o m e t r y will be major m e a n s for protein sequencing in the next decade, their rapid reformation to the ultramicroanalysis m e t h o d s being strongly investigated. For seq u e n c i n g by either method, protein fragmeritation to peptides of appropriate size is essential. A search for and the synthesis of n e w proteases c o m p a r a b l e in specificity with restriction e n d o n u c l e a s e s specific to each of the 20 amino acids is important for protein sequencing and protein engineering in the future.
287
Note a d d e d in p r o o f T h r e e interesting articles and s y m p o s i u m proceedings have recently b e e n published. These deal w i t h electrospray ionization 44 and its c o m b i n a t i o n with ion trap mass s p e c t r o m e t r y 45, and ion trap mass spectrometry alone 46, looking t o w a r d p r o t e i n m i c r o s e q u e n c i n g at s u b p i c o m o l a r concentrations. Advances in sequencing t e c h n i q u e s have also r e c e n t l y been covered in Ref. 47.
References 1 Hugli, T. E. (ed.) (1989) Techniques in Protein Chemistry, Academic Press 2 Matsudaira, P. T. (ed.) (1989) A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press 3 Wittmann-Liebold, B. (ed.) (1989) Methods in Protein Sequence Analysis Springer-Verlag 4 Findley, J. B. C. and Geisow, M. J. (eds) (1989) Protein Sequencing: A Practical Approach, IRL Press 5 Allen, G. (1989) Sequencing of Proteins and Peptides, 2nd edn, Elsevier 6 Shively, J. E., Paxton, R. J. and Lee, J. D. (1989) Trends Biochem. Sci. 14, 246-255 7 Yuen, S. W., Chui, A. H., Wilson, K. J. and Yuan, P. M. (1989) Biotechniques 7, 74-82 8 Tsunasawa, S., Takakura, H. and Sakiyama, F. (1990) J. Protein Chem. 9, 265-266 9 Biemann, K. (1988) Biomed. Environ. Mass Spectrom. 16, 99-111 10 Ganno, S., Wakabayashi, K., Yagi, Y. and Sakiyama, F. (1990) in High Performance Liquid Chromatography of Proteins and Peptides II (Ikenaka, T. and Sakiyama, F., eds), pp. 3-10, Kagaku Dojin (in Japanese) 11 Tous, G. I., Fausnaugh, J. L., Akinyosoye, O., Lackland, H., WinterCash, P., Victorica, F. J. and Stein, S. (1989) Anal. Biochem. 179, 50-55 12 Yu, M. and Dovichi, N. J. (1989) Anal. Chem. 61, 37-40 13 Kennedy, R. T., Oates, M. D., Cooper, B. R., Nickerson, B. and Jorgenson, J. W. (1989) Science 246, 57-63 14 Liu, T-Y. and Boykins, R. A. (1989) Anal. Biochem. 182, 383-387 15 Masaki, T., Fujihashi, T., Nakamura, K. and Soejima, M. (1981) Biochim. Biophys. Acta 660, 51-55 16 Kawata, Y., Sakiyama, F. and Tamaoki, H. (1988) Eur. J. Biochem. 176, 683-697 17 Tsunasawa, S., Masaki, T., Hirose, M., Soejima, M. and Sakiyama, F. (1989) J. Biol. Chem. 264, 3832-3839 18 Maier, G., Drapeau, G. R., Doenges,
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K-H. and Ponstingl, H. (1987) in Methods in Protein Sequence Analysis 1986 (Walsh, K. A., ed.), pp. 335-337, Humana Press Chen, L., Cotter, R. J. and Stults, J. T. (1989) Anal. Biochem. 183, 190-194 Hafok-Peters, Ch., Maurer-Fogy, I. and Schmid, E. R. (1990) Biomed. Environ. Mass Spectrom. 19, 159-163 Caprioli, R. M., Moore, W. T., Dague, B. and Martin, M. (1988) J. Chromatogr. 443, 355-362 Grossmann, P. D., Colburn, J. C., Lauer, H. H., Nielsen, R. G., Riggin, R. M., Sittampalam, G. S. and Rikard, E. C. (1989) Anal. Chem. 61, 1186-1194 Cobb, K. A. and Novotny, M. (1989) Anal. Chem. 61, 2226-2231 Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. and Whitehouse, C. M. (1989) Science 246, 64-71 Aoyama, H., Iwamatsu, A., Dib6, G., Tsunasawa, S. and Sakiyama, F. (1988) J. Protein Chem. 7, 191 Tsugita, A., Arai, I., Kamo, M. and Jone, C. S. (1988) J. Biochem. 103, 399-401 Yamamoto, A., Toda, H. and Sakiyama, F. (1989) J. Biochem. 106, 552-554 Rose, K., Savoy, L. A., Simona, M. G., Offord, R. E. and Wingfield, P. (1988) Biochem. J. 250, 253-259 Knmazaki, T., Fujita, A., Terasawa, K., Shimura, K., Kasai, K. and Ishii, S. (1987) J. Biochem. 102, 1539-1546 Ohuchi, C., Kondo, J. and Hishida, T. (1988) Seikagaku 60, 875 (in Japanese) Miller, C. G., Kong, C. and Shively, J. E. (1989) in Techniques in Protein Chemistry (Hugli, T. E., ed.), pp. 67-78, Academic Press Sun; Y. and Smith, D. L. (1988) Anal. Biochem. 172, 130-138 Rodriguez, H., Nevins, B. and Chakel, J. (1989) in Techniques in Protein Chemistry (Hugli, T. E., ed.), pp. 186-194, Academic Press Maley, F., Trimble, R. B., Tarentino, A. L. and Plummer, T. H. (1989) Anal. Biochem. 180, 195-204 Tomiya, N, Awaya, J., Kurono, M., Endo, S., Arata, Y. and Takahashi, N. (1988) Anal. Biochem. 171, 73-90 Oku, H., Hase, S. and Ikenaka, T. (1990) Anal. Biochem. 185, 331-334 Poulter, L., Earnest, J. P., Stroud, R. M. and Burlingame, A. L. (1988) Biomed. Environ. Mass Spectrom. 16, 25-30 Hunt, D. F., Yates III, J. R., Shabanov, J., Bruns, M. E. and Bruns, D. E. (1989) J. Biol. Chem. 264, 6580-6536 Furuya, M., Akashi, S. and Hirayama, K. (1989) Biochem. Biophys. Res. Commun. 163, 1100-1106
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40 Roepstorff, P. (1989) Acc. Chem. Res. 22,421-427 41 Karas, M., lngendoh, A., Bahr, U. and Hillenkamp, H. (1989) Biomed. Environ. Mass Spectrom. 18,841-843 42 Kawata, Y., Sakiyama, F., Hayashi, F. and Kyogoku, Y. (1990) Eur. J. Biochem. 187, 255-262 []
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Biochemical reactions in supercritical fluids
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--Fig. 1 250
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75°1C~ " j
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Kozo Nakamura
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Supercritical fluids (SCF), which have been gaining recognition as suitable solvents for extraction of biomolecules, also show promise for use as novel media for enzymatic reactions. Their advantages, compared with organic solvents, include, among others, easily controllable solubility of components, high diffusivity, low toxicity and improved reaction rates. The use of SCF should facilitate the integration of biochemical reactions and separation processes.
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All materials have critical points which determine their phase behaviour: the critical temperature (To) is that above which a gas cannot be liquefied by increasing pressure; the critical pressure (Pc) is that at which, at the critical temperature, a liquid may exist in equilibrium with the gas phase. For example, Tc and Pc for carbon dioxide are 31.0°C and 73.8 bar, respectively. Supercritical fluids (SCF) are those which exist as fluids at temperatures and pressures above their critical points. They are of interest in industrial processing since their solvent properties vary considerably with relatively minor changes in temperature and pressure. A recent review by Randolph 1 indicated that the advantages of SCF solvents for extraction could outweigh such disadvantages as lower solubilities and higher capital costs compared with liquid solvents. Many of the properties of SCF which suit them for use in extraction processes also render them attractive as a medium for biocatalytic reactions:
K. Nakamura is at the Department of Biological and Chemical Engineering, Gunma University, Kiryu, Gunma 376, Japan.
• high diffusivity and low surface tension lead to reduced internal mass-transfer limitations for heterogeneous chemical or biochemical catalysts; • the dramatic sensitivity of solubility to changes in pressure and temperature may be used to manipulate the selectivity of a reaction and to recover a product from reactants. The solubility or the state of solute molecules may also be changed with the addition of a small amount of properly chosen co-solvent to control the reaction kinetics; • The pressures associated with suitable SCF such as supercritical CO2 (SCCO2) or ethane are not so high as to damage biopolymers, while the temperatures required are appropriately low for thermally labile biomolecules; • the use of SCF is not accompanied by the problem of solvent residues in the reaction product because SCF solvents are gases under atmospheric conditions. In particular, the low toxicity and reactivity of SCCO2 make it attractive as a non-aqueous reaction medium for food and pharmaceutical products. SCF as media for chemical reactions have been reviewed by
@ 1990, Etsevier Science Publishers Ltd (UK) 0167 - 9430/90/$2.00
Solubility of water in high pressure carbon dioxide: comparison of values derived using Eqn 1 (lines), with experimental data (symbols) obtained by Wiebe et al. 4
--Fig. 2 f6ooc 3O 25
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Solubility of stearic acid in high pressure carbon dioxide, calculated using Eqn 1.
Subramaniam and McHugh 2, with examples of hydrolysis, thermal and photochemical reactions, isomerization and heterogeneous catalytic