developments in protein microsequencing

Adv. Biophys., Vol. 23, pp. 81-113 (1987)

DEVELOPMENTS IN PROTEIN MICROSEQUENCING

AKIRA TSUGITA

L;fe Science Institute, Science University of Tokyo, Noda, Chiba 278, Japan

Sequencing biological macromolecules is to translate text patterns expressed as chemical structures into information that reflects the blueprints of cellular structures and functions. Less than two decades ago the genetic code was established, and since then unbelievable progress has been achieved in the sequencing techniques of DNA. This has resulted in the clarification of more than 20,000 gene structures which has allowed at least as many protein sequences to be deduced from the DNA sequences. The progress in protein sequencing techniques improved remarkably during the period from 1950 until 1965, however, progress since that time has not kept pace with the developments in DNA sequencing. Initially, the characteristics of nucleic acid were thought prohibitive in determining their sequence when compared with those of proteins. Essentially, both proteins and nucleic acids are made of linear polymers; the differences, are in their building blocks and lengths: proteins are composed of twenty different types of amino acids which display a great variety of chemical characteristics and are of relatively short length (average 300 amino acids). However, nucleic acids consist of only four kinds of chemically similar units and are quite long. The difficulty in sequencing long stretches of similar units of nucleic acids turned out to be illusory, whereas the apparent breakdown of the protein sequence proved to be the more difficult task.

81

82 A. TSUGITA

Sanger and Tuppy first established the sequence of insulin in 1951

(2) and during the following 25 years about 600 proteins (80,000 residues)

were sequenced. This number has noticeably increased with recent

developments in protein sequencing techniques (3), such as the use of

automatic protein sequencers (4), gas phase sequencers (5), mass spec-

trometry including field desorption and first atom bombardment tech-

niques (6), fluorescent amino reagents (7), and a variety of applications

of high performance liquid chromatography (HPLC).

In the nucleic acid field there has been a successive development

of ingenious techniques: use of a variety of restriction nucleases, com-

bination of other nucleases and polymerases and DNA sequencing gel

electrophoresis techniques (including terminal labeling and partial ter-

mination in enzymatic polymerization or specific chemical partial de-

gradation). These techniques not only overcame the above mentioned

difficulties thought inherent in nucleic acid structure but were also de-

veloped at a faster rate than protein sequencing techniques.

The techniques of nucleic acid sequencing have become popular

because they are simple, accurate, quick, and sensitive and now an auto-

matic machine is even available. However, there is still a great need for

protein sequencing in order to substantiate the results deduced from the

DNA sequences, including starting and ending positions, reading frames,

intron regions and post-translational processing. Proteins may be com-

pletely sequenced in the classical way in many cases where their coding

DNA is not known. Partial protein sequences are also required for the

synthesis of oligonucleotide for cloning, and for the synthesis of poly-

peptides to produce mono- or polyclonal antibodies.

The present demands on protein sequencing techniques are ob-

vious, they should emulate the achievements of nucleic acid sequencing

by being accurate, sensitive, quick, and simple. This paper is not a general

review of the current techniques which meet these requirements, but

rather is limited to the procedures recently developed in the author’s

laboratory. These may be divided into several segments covering puri-

fication of proteins, determination of amino acid composition, N- and C-

terminal sequencing, terminal labeling, and chemical monitoring of active

regions, each with examples of application.

I. PURIFICATION BY GEL ELECTROPHORESIS AND EXTRACTION

Separation and purification of proteins and peptides in micro scale have

been successfully made by HPLC. The scale has been further reduced

by using microbore HPLC columns. Recovery is one of the serious draw-

DRVELOPMENTS IN PROTEIN MICROSEQUENCING 83

backs with this technique; however, recent developments of column materials have improved this.

Meanwhile, the use of polyacrylamide gel electrophoresis has lent itself to extremely wide applications in the separation and identification of proteins (8). The method is sensitive, efficient, and simple, and the introduction of the silver staining technique has allowed extremely high sensitivity of protein detection (ng or pmol of protein (9)). There are two interesting areas which we have developed: a) method for the simple and effective extraction of proteins from conventional gels allowing further protein sequence studies, and b) an extension of the gel electrophoresis technique, which is especially useful for separating small peptides (less than 10,000 molecular weight) using a volatile buffer system.

1. Extraction from Conventional Polyacrylamide Gel (7 7, 72) There are many established extraction procedures of proteins and peptides from polyacrylamide gels including electroelution and electroblotting. Some of our experiences with electroelution have shown that it allows protease digestion even in the presence of a detergent, such as SDS or proteinase inhibitors, especially when the sample is crude, such as for cell extracts. Electroblotting on a glass filter paper previously coated with polybrene or another polymer has recently been found to be quite practical, particularly for sequencing protein when used in a gas-phase sequencer. Detergents or ampholine, which is in the medium of electrophoresis or electrofocusing, is difficult to remove completely and if removed the protein often loses its solubility. We found that 70% formic acid was effective to dissociate proteins from these reagents, as well as to extract proteins from the polyacrylamide gel.

After electrophoresis the gel is stained by the Coomassie Blue method (without staining when protein is radioactively labeled) and the spot (in two-dimensional gels) or band (in one-dimensional gels) is cut out and placed in more than 4 times its volume of 70% formic acid. The gel usually swells to 2.5 times its original volume after equilibration in 70% formic acid. Extraction is carried out by gently shaking the gel (as with DNA extraction) at room temperature for 16-24 hr depending on the molecular sizes of the proteins or peptides. The color is a minimum in- dicator of the equilibration. This extraction is repeated 2 to 3 times. The extracted solution is evaporated using a rotary evaporator after adding a drop of octyl alcohol to avoid foaming. The residue is dissolved in a small amount (lo-300~1) of 70% formic acid and applied to a molecular sieving column (various sizes from 0.4x4 cm to 0.9~ 80 cm) of Biogel PlO (100-200 mesh) which has been equilibrated with the same acid.

84 A. TSUGITA

The protein is eluted first from the column separated from other small

molecules including detergents, electrophoresis buffers, glycine and blue

dye. The eluted protein in 70% formic acid is ready to use for various

protein characterizations such as amino acid composition, sequencing,

and enzymatic digestion. The extracted protein is usually tested for homo-

geneity by small scale gel electrophoresis.

There is some suspicion that the 70% formic acid cleaves peptide

bonds in proteins. However, it should be noted that formic acid (66-80%)

has been widely used as a solvent in the cyanogen bromide reaction for

specific cleavage of the methionine peptide bond. In most cases, no cleav-

age of peptide bonds has been observed. There is one exception, the

aspartyl-prolyl bond is partially cleaved by 70% formic acid at 37°C. It

is wise, therefore, to test for this by preliminarily incubating an aliquot

of protein in 70% formic acid at 37°C for 24-48 hr, followed by SDS-

gel electrophoresis and silver staining.

The recovery with this method has been observed to be above 60%

and in many cases as high as 95% using 5-100 pg of protein (13, 74).

In order to achieve high recovery, cutting out the gel without either crush-

ing it or further dividing it into small pieces is most important. Although

some publications recommend crushing the gel, small gel pieces reabsorb

a large portion of the protein in a limited volume of formic acid. There-

fore, after each extraction, filtration of the solution with a small glass

filter is recommended. In addition, gels more than 1 mm thick need a

long time (more than 24 hr) for extraction. For very small amounts of

protein, it is recommended that equipment such as columns, tubes and

filtering tools be siliconized.

From our experience we believe that this extraction method has the

following advantages: virtually all proteolysis is avoided, exact bands can

be cut out, small undesired molecules including SDS are removed, and

after extraction the protein is ready for further experiments. The above

procedure has successfully been employed for a variety of proteins; the

partial degradation products of ATPase inhibitor (IFI), using 1 nmol,

5 steps of the N-terminal sequence, 9 residues of the C-terminal sequence

with carboxypeptidase digestion and the amino acid composition have

been determined (11); by proteolytic partial degradation, 14 peptides of

an Escherichia coli membrane protein, R-phage receptor, were isolated and

several steps of the sequences from the N- and the C-termini and their

compositions were demonstrated, which contributed to the topology of

this protein (15); for a subfraction of human Hl histone, three different

types of molecular species were deduced from their compositions and

DEVELOPMENTS IN PROTEIN MICROSEQUENCING 85

C-terminal analyses (73, 74); f or a partial degradation product of adeno- virus DNA binding protein (45kD), the N- and C-terminal sequences and the composition were used to determine the location of the cleavage sites in the protein sequence (76). Other examples of purification and charac- terization of proteins are given in the reference section (77-79).

2. A New Polyacrylamide Gel Electrophoresis System for Small Peptides

(20) In general, separation and purification of small proteins and peptides are currently achievable by HPLC. However, adequate recovery and solubility of samples in solvent systems are still problematic. Small peptides have proved difficult to separate by gel electrophoresis due to their inability to form stable SDS complexes and to being diffused out from the gel by the fixing, staining and destaining procedures. To overcome these, we have developed a new gel electrophoresis system to facilitate separations of peptides and proteins ranging in molecular weight from 200 up to 100,000 (20). This system involves covalently binding the NH, reagent, 1,3,6-trisulf- onylpyrene 8-isothiocyanate to the protein. By modification of this reagent, the addition of strongly negative charges to proteins is achieved which allows migration according to molecular weight in an electric field. The modified proteins, being fluorescent require neither fixation nor staining for their detection. An additional advantage of such modification is the increased solubility of the protein which makes its extraction more efficient. The modified proteins can be applied to either the conventional Tris HCI-glycine electrophoresis buffer system (8) or to the new buffer system consisting of a volatile buffer, triethyl amine formic acid pH 11.7. In the conventional buffer system, the extraction may follow the procedure described in the previous section including gel filtration (77, 72). This can only be applied to peptides whose molecular weight is more than 10,000 because of the difficulty in separating the peptides from small molecules, and the NH, terminal residue of the peptide is lost because the modified N-terminal amino acid is cyclized off in 70% formic acid. In the first system, however, protein is not cleaved from the N-terminus and is easily extracted with 50% pyridine. Peptides are freed from salts and other small molecules simply by evaporation. Peptides extracted by the above method have been used for the following sequence analysis.

Samples are dried in a 400 ,~l Ravin reaction tube, then trisulfonyl- pyrene isothiocyanate is added (Serva, 40 mg/ml of formic acid) and dried. Twenty to forty-five microliters of 0.15 M dimethylallylamine / trifluoroacetic buffer pH 9.5 (containing 10% dioxan and 1% Triton X-

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DEVRL.OPMENTS IN PROTEIN MICROSEQURNCING 87

0.5 1.0 0.5 1.0

Relative distance Reldtive distance

Fig. 1. Electrophoresis patterns of peptides and proteins.

Upper part: fluorescence patterns of the electrophoresis gels. Gels were 13.5

cm wide, 0.15 cm thick. The lower was 8 cm long and the upper 3 cm. Sam-

ples, from right to left with M, in parentheses, are listed below. The upper

arrows show the top of the lower gel and the lower arrows the migration front.

a: 32% acrylamide, 27.5 W, 320-420 V, 110min. Samples Phe-Ala (236),

Leu-Trp (317), Leu-Trp-Met (449), Alas-Pro-Tyr-Alas (705), angiotensin II

(1,046), angiotensin I (1,297), glucagon (3,483). b: 20% acrylamide 30 W,

310-380 V, 60 min. Alas-Pro-Tyr-Alas (705), angiotensin II (1,046), glucagon

(3,483), adrenocorticotrophic hormone fragment (18-39) (2,466), aprotinin

(6,500), lysozyme (14,400). c: 10% acrylamide 35 W, 300-330 V, 40min,

glucagon (3,483), aprotinin (6,500), lysozyme (14,400), myoglobin (17,200).

ovalburnin (45,000). d: 5% a&amide 40 W, 118-116 mA, 320-300 V, 40

min, myoglobm (17,200), chymotrypsinogen A (25,000), ovalbumin (45,000),

albumin (68,000), phosphorylase B (92,500), catalase (60,000).

Lower part: showing linearity between molecular weight and migration dis-

tance in the upper gels. 0 molecular weight; l corrected molecular weight after modification (75).

88 A. TSUGITA

Fig. 2. Electrophoresis patterns of human angiotensins. 30% acrylamide

25 W, 310-380 V, 70 min. Samples, from left to right, C-peptide (3,617)+

glucagon (3,483), angiotensin I (1,297), angiotensin II (1,046), angiotensin

III (931). The last sample, angiotensin III which loses 1 mol of aspartic acid

from angiotensin II, migrates a short distance due to charge difference.

The upper white mark show-s the top of the lower gel and the lower large

spots indicate the migration front (the unreacted reagent).

100) is added to the reaction mixture which is then transferred into a small glass tube (50 111 volume) and sealed under N, gas (for details see ref. 20). The reaction is carried out at 100°C for 15 min.

The composition of the volatile buffer system is listed in Table I. The electroconductivities of the buffers should be strictly monitored because these values vary depending upon the reagents used and subse- quently disturb the electrophoresis results. High pH buffers (7, 71) are used to avoid the disturbance of electrophoretic mobility caused by the guanidino group of arginine. The acrylamide/bisacrylamide solution is perferentially passed through Dowex 1 x2 (acetate form) in order to eliminate acrylic acid contamination. Electrophoresis is carried out with a relatively high constant power, with ice water cooling the plate, and with the electrode buffers circulating due to the absence of the zwitterion compound, glycine. Typical examples of the electrophoresis patterns are shown in Fig. 1. The extraction is made by cutting out the bands and soaking them in 10 times their volume of 50% pyridine. Figure 2 shows one of the applications of this method; a simple separation of human angiotensins (A. Tsugita, unpublished data). The recovery of the entire procedure until extraction is usually around 90% for glucagon, insulin /3 chain. When the extracted sample is subjected to Edman degradation, the cyclization recovery is quantitative at 50°C for 10 min with trifluoroacetic acid (TFA).

DJWELOPMENTS IN PROTEIN MICROSEQUENCING 89

II. RAPID AND MICRO METHODS FOR HYDROLYSIS OF PROTEINS (ZZ-

24

One of the classic but still important characterizations of a protein is its amino acid composition. The recent needs are for both rapid and micro- scale protein hydrolysis and a method for the complete hydrolysis of hydrophobic proteins.

Developmental progress of automatic amino acid analysers equipped with post column ninhydrin reaction systems enable realisation of a sensitivity of 100 pmol to 2.0 run01 amino acid in full scale. HPLC and pre- or post-column derivatization with phenylisocyanate or with various fluorescent and diazo reagents have achieved pmol level analysis. Hydrolysis of proteins into amino acids has been carried out by treatment with acid, alkali or enzymes. The most popular method has been acid hydrolysis with constant boiling HCI (5.7 M) at 106°C for more than 24 hr (27). Elevation of the temperature accelerated the rate of peptide bond hydrolysis as shown in curve A in Fig. 3. A temperature elevation of 60°C may accelerate hydrolysis by 28=64 times, thus reducing hydrolysis times from hours to only minutes. However, curve A shows considerably less acceleration effect than expected. Addition of TFA and elevation of the molarity of HCl are needed to satisfy the requirements for such short

loo-

80-

i10 130 150 170 190 210

Temp. (‘C)

Fig. 3. Efficiency of hydrolysis at various temperatures. Val-Glu (3 nmol)

was hydrolysed with 6 M HCl (curve A), TFA: HCI (1: 2) (curve B) and

vapour of 7 M HCl and 10% TFA (curve C) at various temperatures. The

vertical axis is the percentage of yields of glutamic acid analysed with an

amino acid analyser.

90 A. TSUGITA

hydrolysis times. TFA is a strong organic acid having a pK, 0.23, a high

vapour pressure and low boiling point (72.5%) ; the use of such an organic

acid may allow the acid to become accessible to the hydrophobic region

of the peptide. A new rapid hydrolysis method has been proposed based

on the above observations (22-24).

In the attempts being made to significantly reduce the amount of

protein required to determine the amino acid composition, we met serious

problems of contamination during the process of hydrolysis and/or the

following procedure of amino acid analysis. This contamination was ob-

served both with the conventional 6 M HCI method and our new methods.

The hydrolysis acid contains amino acids or peptides which, upon hy-

drolysis produce amino acids. (The contamination may also come from

dust in the air or on such equipment as pipettes, tubes, etc.). To deter-

mine the minimum volume of hydrolysis reagent required, 5 /*g of protein

were hydrolyzed with various volumes of acid in 0.8 mm x 8 cm test tubes.

Experiments showed that 5-10 pd of acid was sufficient to hydrolyze 5 /lg

of protein. The following tryptophan analysis was developed on the basis

of this observation.

Tryptophan is almost completely decomposed under the conditions

generally employed for acid hydrolysis. To circumvent this decomposi-

tion, several methods including spectrophotometry (25), basic hydrolysis

(26), and acid hydrolysis under reducing conditions (27, 28) have been

proposed. Either these methods are not satisfactory or they consume

relatively large amounts of protein. Consequently, people have neglected

the determination of tryptophan contents. By minimizing a hydrolyzing

reagent, mercaptoethanesulfonic acid, and by raising the temperature, a

micro and rapid hydrolysis method for tryptophan was proposed (2s).

When vapour of the acid was used, hydrolysis was still achieved

and, as expected, the contamination was considerably reduced. The

extent of hydrolysis is more efficient than that obtained by the liquid

method (Fig. 3, curve C). Vapour phase hydrolysis of protein at an ele-

vated temperature has been developed.

In the following steps of amino acid analysis it is important to mini-

mize the contamination involved in the sampling buffer and the chroma-

tography buffers in the amino acid analysis system. (Most of the conventional amino acid analysers or post column HPLC systems use citrate or

borate buffers for the ion exchange column). The use of 0.01 M HC1

(from doubly distilled HCl) instead of the sample buffer (0.2 M citrate

buffer pH 2.2, Pierce) reduced the amino acid contamination to at least

l/50. Recently Pierce changed the chromatography system from citrate

DRVELOPMRNTS IN PROTEIN MICROSEQUENCING 91

to a mineral buffer system which considerably reduced the contamination level of the amino acids.

1. Rapid Hydrolysis with a Mixture of TFA and HCI (22-24)

Protein (1-5 pg) was dissolved in 50 ,~l of a mixture of TFA and HCl (1: 2 v/v) in a test tube (0.8 x 8 cm). The tube was vacuum sealed in ice and hydrolysis was carried out at 166°C for 25 and 50 min. The hydrolysate was evaporated with a rotary evaporator at 60°C. Then the residue was dissolved in 0.01 M HCl and applied to an amino acid analyser. When sealing with a mechanical vacuum pump TFA perferentially evaporates at room temperatures even within 30 set which causes insufficient hydrolysis. The use of ice to cool the hydrolysis tube and evacuation for less than 3 min is recommended to prevent the evaporation of TFA. TFA in the hydrolysis acid mixture is a high grade reagent (Fluka) but is not sequanal grade because of its oxidation of amino acids. Oxidation of methionine is prevented by adequate evacuation. The addition of 0.01% phenol to the hydrolysis acid prevents chlorination of the tyrosine residue. A recent publication (24) lists the decomposition extent of uncommon amino acids and widely used derivatives of amino acids. The method is

TABLE II

Recovery of Amino Acids after Incubation (a) at 166°C by Liquid Phase TFA: HCl

(1: 2) and (b) at 158°C by Vapour Phase 7M HCL 10% TFA

Liquid phase (a) Gas phase (b)

25 min 50 min 22.5 min 4s min

ASP 94 97 99 103

Thr 89 82 92 84

Ser 75 61 85 78

Glu 100 100 100 100

Pro 84 85 96 98

Gly 93 91 104 108

Ala 113 110 115 101

Val 102 102 98 98

Met 70 65 87 76

Be 97 99 93 95

Leu 96 100 92 94

T yr 92 94 89 81

Phe 99 101 90 87

His 106 101 96 99

Lys 101 101 95 98

Arg 108 106 100 100

Recoveries are expressed as a percentage of the value of glutamic acid taken as 100%.

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0 2.

8 2.

7 2.

6 3.

1 2.

8 3

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6 1.

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8.1

7.8

7.7

8.6

8.7

9 3

6.4

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6.4

6.1

7 6.

9 5.

9 4.

3 7.

7 5.

5 8-

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20

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20.7

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His

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Arg

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ecov

ery

1.1

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40

l-2

3.7

3.9

4.5

2.5

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94 A. TSUGITA

simple, convenient and recovers more amino acid from the hydrophobic

protein than the 6 M HCI method (22,23). The application of this methods

has been successful for proteins and peptides (17, 13-19, 30-32, 47-51).

Table II lists the recoveries of amino acids under the above conditions

and Table III some applications of this method to proteins.

2. Vapour Hydrolysis (33) Protein (0.1-5 pg) is placed in a small test tube (0.4~ 4 cm) and eva-

porated. The small test tube is placed in a larger test tube (1.3 x 10 cm)

which contains 100-300 111 of 7~ HCl, 10% TFA and 0.1% phenol. The

proportions of these hydrolysis reagents were varied and tested for the

efficiency of hydrolysis and decomposition of amino acids. Figure 3,

curve C shows the efficiency of hydrolysis at various temperatures. The

tube is cooled in an ice bath evacuated for 1 min at 0°C and vacuum

sealed with a flame. Hydrolysis is carried out at 158°C for 22.5 to 50 min.

After hydrolysis the small test tube is taken out and placed in a vacuum

desiccator to remove any traces of acid. The residue is dissolved in 0.01 M

TABLE IV Amino Acid Composition of Protein by the Present and Conventional Methods&

Glucagon Cytochrome c

from by hydrolysis for from by hydrolysis for

sequence 22.5, 45’ 24hr 72hr sequence 22.5’ 45’ 24hr 72hr

Asp 4 4.1 4.2 4.0 4.4 8 8.1 8.3 7.8 7.7

Thr 3 2.8 2.7 3.0 2.9 10 9.0 8.1 8.4 8.1

Ser 4 3.5 3.0 3.7 3.9 0

Glu 3 3.0 3.0 3.0 3.0 12 11.9 12.0 10.9 11.2

Pro 0 0 4 3.8 3.8 3.8 3.6

GUY 1 1.1 1.1 1.3 1.5 12 11.8 12.0 10.9 11.2

Ala 1 1.1 1.1 1.0 1.4 6 6.0 6.0 6.0 6.0

CYS 0 0 2 1.1 0.8 0.0 0.0

Val 1 1.1 1.2 1.0 1.1 3 2.6 3.0 2.9 3.1

Met 1 0.6 0.6 0.2 0.4 2 1.9 1.8 1.3 1.1

Ile 0 0 6 4.7 5.5 4.4 5.5

Leu 2 1.9 1.9 2.0 1.8 6 5.4 5.6 4.8 5.7

Tyr 2 1.6 1.6 1.9 0.8 4 3.4 3.5 2.8 2.3

Phe 2 1.7 1.8 1.9 1.4 4 3.6 3.6 3.2 3.0

LYS 1 1.0 1.1 0.9 1.1 19 18.8 18.9 16.8 16.7

His 1 1.0 1.0 0.8 1.0 3 3.1 3.0 3.0 2.6

Arg 2 1.9 1.9 2.0 1.8 2 2.1 2.1 1.9 1.9

Trp 1 ND ND ND ND 0 ND ND ND ND

8 The 22.5 min and 50 mm hydrolyses were with 7M HCl and loo/, TFA at 158°C

(vapour phase); the 24hr and 72hr hydrolyses were with 6~ HCl at 107°C (liquid phase).

DRVRLOPMENTS IN PROTEIN MICROSEQUENCING 95

HCl and applied to an amino acid analyser. Instead of the large test tube, more convenient reaction apparatus such as the Pierce reaction tube has been tested but without success. It has been observed that the combination of both high temperature and the aggressiveness of the acid mixture occasionally broke the vacuum and caused oxidative decomposition of serine, theronine, and other amino acids. This method reduces the contamination of the amino acids considerably (less than 2 pmol tested by o-phtalaldehyde method (33)). Development of a method to avoid contamination such as the one mentioned above is required for sensitive amino acid analysis at the level of less than 100 pmol full scale (corresponding to 100 ng-1 pg of protein).

Decomposition of amino acids and applications for proteins are listed in Tables II-IV under the present method followed by analysis with a Durum D500 equipped to a sensitivity of 2.5 nmol or 500 pmol of amino acid full scale (ninhydrin detection system). The method provides sufficient hydrolysis but decomposition of threonine and serine is found to slightly exceed that of liquid phase hydrolysis (22, 23). This method

from sequence

Myoglobin CGMMV coat protein

by hydrolysis for from by hydrolysis for

22.5’ 45’ 24hr 72hr sequence 22.5’ 45/ 24hr 72hr

8

5

6

19

4

11

17

0

8

2

9

18

3

6

19

12

4

2

8.2 8.3 8.4 8.1

4.8 4.5 4.9 4.8

5.4 4.8 5.8 5.7

18.8 18.9 19.5 18.9

4.2 4.2 3.9 3.9

10.9 11.0 11.6 11.0

17 17 17 17 - - - -

7.6 7.9 4.8 7.3

1.7 1.9 1.8 1.9

6.8 8.4 5.3 7.7

16.1 17.3 15.3 16.3

2.0 0.5 2.0 2.0

5.6 5.9 4.6 4.9

18.3 18.7 17.1 19.0

11.8 11.8 10.7 11.8

4.0 4.0 3.5 4.1

ND ND ND ND

18 18.0 18.1 18.1 17.2

12 10.8 10.0 12.0 10.9

22 18.4 17.0 20.8 19.6

10 10.0 10.1 10.3 9.7

9 8.5 8.6 7.9 7.8

5 5.0 5.7 6.4 5.9

20 20 20 20 20

0 ----

14 12.7 14.0 11.4 11.7

0

8 7.1 7.6 7.0 7.1

12 11.2 11.3 11.2 9.8

4 3.2 2.8 2.4 1.4

11 9.8 9.5 9.1 7.8

4 3.5 3.6 3.6 3.6

0

10 9.6 9.5 9.8 8.5

1 ND ND ND ND

Values were based on the value for alanine, except for glucagon which was based on the

value for glutamic acid.

96 A. TSUGITA

was found to be especially powerful when the protein is extremely hydrophobic such as the proteolipid protein of myelin (19). The method was applied to several proteins.

3. Tryptophan Microanalysis (29)

One to five micrograms of protein was placed and evaporated in a tube (hand-made, volume 100 ~1). Thirty microliters of 3 M mercaptoethanesulfonic acid was added and the tube evacuated and sealed. Hydrolysis was carried out at 166°C for 2.5 min. After cooling, 50 ,ol of 1.38 M NaOH was added to the hydrolysate which was then subjected to analysis. The analysis equipment was composed of a single column system (1.75 x 500 mm) with a flow rate of 10 ml/min at 50°C. The pH of the buffers was 3.25 (O.~M), 4.25 (O.~M), and 5.28 (1.1 M). Recovery of amino acids (Table V, column a) is reasonable including those of tryptophan (93%), threonine, and serine. The recovery of proline appears to exceed loo%, which is due to its coelution with the product formed between cysteine and mercaptoethanesulfonic acid through a disulfide bridge. Table V,

TABLE V Recovery of Free Amino Acids and Composition of Lysozyme

Free amino acid

a b

Lysozyme

Amino acid Recovery

(%) Observed (mol/mol)

(corrected) Theoretical

ASP Thr

Ser

Glu

Pro

Gly Ala

Val

Met

Ile

Leu

TYr Phe

His

LYS

Arg

Trp

CYS

95.2 21.7

91.4 6.4

86.0 8.6

100.0 5.0

145.7 2.9

100.0 12.6

100.0 12.6

97.0 4.9

110.0 2.2

91.0 4.6

100.0 8.0

98.8 2.7

100.0 3.0

100.0 1.0

98.7 5.5

100.0 10.5

93.1 5.4 - -

21

(7.0) 7

(10.0) 10

5

2

12

12

6

2

(5.5) 6

8

3

3

1

6

11

(5.8) 6

8

DEVJZLOPMENTS IN PROTEIN MICROSEQUENCING 97

column b, shows the composition of egg white lysozyme. The unknown proteins T4 ligase (30), EcuRV nuclease and EcoRV methylase (37) were also analysed. The numbers of tryptophan residues have been confirmed in all cases by DNA sequencing of their genes.

III. N-TERMINAL SEQUENCING

One of the most pressing needs of protein sequencing is the reduction of the amount of protein required.

For N-terminal sequencing, micro scale Edman degradation is used exclusively. The spinning cup method (Beckman) is able to analyse 100 pmol of protein and the repetitive yield is relatively high (95-99x) ; consequently, a relatively high number of sequence steps are achieved. How- ever, the maintenance of the machine to keep this high repetitive yield is difficult. We first demonstrated semi-nanomole (300 pmol) sequencing of E. coli cyclic AMP receptor protein (32) and recently about 30-50 pmol of several other proteins for 15-20 steps.

The gas phase sequencer (Applied Biosystems) has provided protein sequences with 50-100 pmol protein (5). The advantage of this machine is its relatively easy handling, hence its popularity in recent years. The disadvantage is the relatively low repetitive yield (90-95x) which may limit the number of sequence steps. In both methods the ultimate limitation is the detection of the final product. The conventional method de- pends on ultraviolet absorbance of the phenylisothiohyantoin derivative of amino acids in HPLC. A highly sensitive detector can analyse a few pmol. 32S-phenylisothiocyanate (PITC) used with a solid phase LKB sequencer (34) and 1251-PITC (35) have been used to increase the sensitivity of detection. Radiodegradation, especially of a%-PITC, affects the main Edman degradation reaction, resulting in the limitation of repetitive yield. Isothioderivatives of fluorescent or diazo compounds have been developed for the sensitization of Edman degradation (52, 53). These methods suffered from problems of poor yields in coupling or cyclization. To break through the wall of pmol detection in Edman degradation we must find another method for sensitive detection of the product. Applica- tion of mass spectrophotometry was suggested (5) but has not yet been realized.

1. A Sensitive Detection Methodfor Edman Degradation (33) We have recently been developing a sensitive detection method for Edman degradation. As in the well established Edman degradation, the classical method of coupling with PITC and the cyclization reaction are con-

98 A. TSUGITA

FL RZ N=C=S + NH2-CH-CO-NH-&H-CO-....

PITC

s PTC peptide

N c- 0 NH-;--; Remaining peptide

I ATZ

RI NeNH

@NH-~-NH-LH-C~-NH-CH~CH~_~+

s Detectable 200f-lf I

Phenylthiahydantain

Detectable 2p

Fig. 4. Reaction mechanism of the sensitization of Edman degradation

intermediates with primary amines. Normal Edman degradation is followed

until the AT2 intermediate is obtained from the N-terminal of the peptide

or protein. Instead of the usual PTH product being made, the ATZ is reacted

with either a radioactive or fluorescent primary amine to produce a sensitized

product. Shown is the radioactive reagent iz51-iodohistamine. Theoretical

detection of this product is between 200-l fmol.

served; however, the resulting anilinothiazolinone (ATZ) derivative is subjected to a sensitizing modification. Instead of conversion to the phenylthiohydantoin derivative, the reaction between the ATZ derivative with amino compounds such as %I-iodohistamine or 9-aminofluorine results in the phenylthiocarbamyl (PTC) derivative as shown in Fig. 4. Figure 5 shows one of the typical reactions between the ATZ derivative of leucine (peak a) and iodohistamine on HPLC. The reaction was followed by an increase of the PTC derivative (peak c) and was completed after 1 hr. The PTC derivative product has been confirmed by NMR and elementary analysis. The yield of recrystallized product was 80% for leucine (suggesting the recovery is almost quantitative). These derivatives of amino acids are stable and can be analysed on silica gel thin layer chromatography and by reverse phase HPLC. The theoretical sensitivity of this method with iodohistamine is 0.1-l fmol with 1261-iodohistamine


C

b)

-L Fig. 5. Time course of the reaction of ATZ Leu with iodohistamine. To a

solid of ATZ Leu (70 ng, 0.28 nrnol) was added 5-iodohistamine hydro-

chloride (200 ng, 0.6 nmol) in 30% pyridine/dimethyl formamide at room

temperature. The solution was heated at 50°C for 1 hr with stirring. The

HPLC chromatograms show the course of the reaction; A, 0 min, room tem-

perature; B, 30 min, 50°C; C, 60 min, 50°C. The HPLC column was a Ser-

vachrom s 100; polyol; PP18 5 pm, 250 mmx4.6 mm. The HPLC system

was isocratic with solvent A being 0.015 M sodium acetate, pH 5 and solvent

B an 80% acetonitrile, 20% methanol mixture. The ratio of solvent A: B was

1: 1.5, respectively. Detection was by optical density at A,,,. The flow rate

was 1 ml/min. An increase in product formation (b) with a corresponding

decrease ATZ (a) concentration over the incubation period can be seen.

and 10-100 fmol with fluorescent NH, compounds, 4-aminofluorecein. Figure 6 shows an HPLC pattern of 4 aminofluorescein derivatives of almost all amino acid AT2 derivatives. The reaction conditions are 50°C for 10 min with 5 times excess of 4-aminofluorescein in dimethyl formamide; routinely by this method 100 fmol sequencing has been achieved in the gas phase sequencer (36). This is also a novel method for sequencing protein in the presence of nucleic acid or in a protein-nucleic acid complex (37).

2. N-terminal Specific Labeling Method (30, 31) Specific labeling of the N-terminus of proteins or peptides is needed for several purposes. Sanger’s DNP method or dancylation method reacts with both N-terminal and &-lysine NH,-residues. It was a breakthrough

100 A. TSUGITA

Fig. 6. Chromatogram of the HPLC separation of fluorescent sensitized products. The HPLC gradient was 0 min, 460,6B; SOmin, 58%B; 52 min, 460/,B where solvent A was 60 mM sodium acetate pH 4.9 and solvent B was methanol. The flow rate was 1 ml/min. The column was a Microsorb C18, 4.6 mm~250 mm. Detection was by fluorescence, with an excitation wavelength of 500 mm and emission wavelength 516 mm. The reaction conditions for the fluorescent primary amine, 4 amino fluorescein was 100 pmol ATZ amino acid with 500 pmol 4 amino fluorescein in 10 ~1 176 pyridine in dimethyl formamide at 50°C for 10 min.

TABLE VI Subunit Molecular Weights Determined with the Present Method Using PITC and S-PITC

Protein S-PITC PITC lLIolecular weight

Insulin 5,540 5,770 5,740 (seq.) (bovine pancreas)

Myoglobin 16,100 17,200 17,200 (seq.) (whale sperm)

Lysozyme 13,500 - 14,300 (seq.) (chicken egg white)

gp22 24,900 - 27,500 (gel)

(T4 phage) Cytochrome c, 25,400 - 32,000 (gel)

(Neurospora) ____~

The residual weights of tryptophan and cysteine were added to all proteins except gp22, for which no analysis for tryptophan and cysteine was carried out. Reference molecular weights were obtained from either sequences (seq.) or gel electrophoresis (gel).

in that it furnished confirmation of a blocked N-terminus, provided the minimum molecular weight of the protein (38), and demonstrated a suc-

cessful protein sequencing strategy (39).

DEVELOPMENTS IN PROTEIN MICROSEQURNCING 101

The principle of this method is that the protein is completely modified with isothiocyanate compounds such as PITC or sulfonated PITC under the conventional Edman conditions. The reagent is removed by extraction, dialysis or column chromatography. The modified protein is subjected to Edman’s cyclization conditions (TFA, at 50°C for 3 min). The first N-terminal residue is cleaved off, simultaneously the NH,-group of the 2nd residue appears and the NH,-groups stay in their modified state. The second coupling reaction is carried out with different NH,- reagents from the first reagents such as W-PITC or dansylchloride. After the excess reagent is removed, the protein is modified, especially at the 2nd N-terminal NH,-group. Table VI shows the minimum molecular weight determined by the above N-terminal labeling method. The data clearly indicate how specific the N-terminal modification has been made

(30

IV. CARBOXY-TERMINAL SEQUENCING

C-terminal sequencing of protein is highly important for justification of cDNA cloning, and for cloning of a large protein gene or a eukaryotic gene where a silent locus is often found.

Tritium specific substitution of the C-terminal carboxy-group (40) is one of the best approaches in combination with the SH-labeled C- terminal peptide separation such as on an anhydrous trypsin column (47).

The other technique, and the conventional one, is the use of carboxypeptidase digestion. There are several problems in obtaining reliable results both for enzymes and protein-substrates. The enzyme has to be completely free from endopeptidase activity since there are often extremely protease-sensitive peptide bonds within polypeptide chains. We have been using three carboxypeptidases, i.e., A, B, and P, whose specificities differ from each other. Carboxypeptidases A and B, having a common optimal pH, and can be used either alone, when they react in a restrictive way, or together. It is recommendable to use diisopropylphosphate treated carboxypeptidases A and B (Sigma) because differently treated (PMSF) enzymes occasionally show endopeptidase activity. Carboxypeptidase Y and carboxypeptidase P at pH 5.5 also show endopeptidase activity but carboxypeptidase P at pH 2.5 can be safely used because little endopeptidase activity has been observed at this low pH. The composition of the volatile buffers (collidine-pyridine-acetate or formate) and enzyme- digestion specificities are listed in a previous publication (42).

The carboxypeptidase method has several disadvantages besides those of endopeptidase contamination, the non-linear reaction kinetics

102 ii. TSUGITA

causes difficulties in ordering the sequence or an enzyme does not cleave specific peptide bonds or specific regions. Therefore, we use combinations of enzymes which compensate for each other, though some specific peptide bonds such as a glycine or a serine cluster cannot be digested by the present enzyme combinations. Moreover, tertiary structures in the substrate resist digestion by enzymes and hydrophobic proteins and are not soluble in the digestion buffers. Substrates have usually been heated to 100°C to denature them and to increase their solubility. Analysis after digestion is carried out for the liberated amino acids with an amino acid analyser, where ninhydrin has 100 pmol sensitivity and o-phtalaldehyde in post column analysis has a 50 pmol range.

We have initiated the use of field desorption (FD)- and fast atom bombardment (FAB) mass spectrometry for detection of the residual peptides after digestion (43-45). The limitation of this method is the size of peptide. Peptides containing more than 50 amino acids are practically unmeasurable in our machine. The following techniques have been developed to overcome some of these difficulties.

I. Field Desorption Mass Spectrometry on Exopeptidase Digestion (43, 44) As previously discussed exopeptidase digestion is not reliable due to the inevitable contamination by endopeptidases. This contamination may lead to a misreading of the terminal sequence, and non-linear kinetics and repetitive amino acid residues in a sequence may limit the application for sequencing. An attempt to overcome these difficulties has been made by using FD mass spectrometry to analyse peptide mixtures obtained from exopeptidase digestion (43, 45).

The digestion is made under the usual volatile conditions in collidine-pyridine-acetic acid-formic acid buffers for different time periods and/or at different temperatures. NH,-buffer and Na-buffers cause con- fusion because of NH,+ or Na+ ions in FD mass spectroscopy. The reaction mixture is dried under vacuum (not over any drying salts such as NaOH or CaCl,). The residue is dissolved in a small volume (1 ,~l each) of water and/or acetic acid and is loaded on the FD emitter. After evaporation of the solvent, the emitter is introduced in the ion source of FD mass spectrometry. Typical results are shown in Fig. 7, which shows a mass spectrum obtained from a mixture of digestions of Leu-Trp-Met- Arg-Phe-Ala with carboxypeptidase A for 1 hr and with carboxypeptidases A and B for 1 hr and 6 hr, all at 37°C. Here, we can observe molecular cations [MH]+ corresponding to the residual peptide digested from the C-terminus. This type of work is not limited to C-terminal determination but is also possible for N-terminal determination using exopeptidases such


824

100 200 300 400 500 600 700 800 900

Corboxypeptidase-degradation mixture Solvent : t-&O/acetic acid

Fig. 7. FD mass spectrum of an aliquot obtained from partial digestion of

Leu-Trp-Met-Arg-Phe-Ala in 0.1 M pyridine-collidine-acetate buffer with

carboxypeptidase(s) at 37°C. An equivalent volume mixture of digests car-

boxypeptidase A for 1 hr, carboxypeptidase A+B for 1 hr and carboxy-

peptidase A+B for 6 hr was analysed (35).

as aminopeptidases. Figure 8 shows aminopeptidase M digestion of the same peptide under different digestion conditions.

These observations clearly show that FD mass spectrometry is a powerful tool for analysing exopeptidase digests. Its advantages are that the experiment shows immediately the endopeptidase digestion products and allows analysis of non-linear kinetics. Its disadvantages are: we can at present only apply this technique to peptides whose molecular weights are less than 6,000; the sensitivity is theoretically about 100 pmol but in practice it consumes more material and the application of samples is not automatic. These problems will soon be solved with the development of the technology needed to increase sensitivity and to facilitate automated sample application. For example, tandem and plasma desorption mass spectrometry has already moved the limitation of molecular mass to 12.5 Kd. Similar work has also been performed independently by other groups (4.5) but only for carboxypeptidase digestion. The method was used for the C-terminal sequencing of the peptide fragments of an unknown protein (44).

2. The Use of Detergents or Alcohols in Proteolytic Digestion (33, 57) The important conditions for proteolytic digestion are that the substrate

104 A. TSUGITA

‘iii/ a;7 / ir,:: .,;;, , , , 500 550 600 650 700750 800 850 900950 1000

Leu-Trp-Met-Arg-Phe-Ala aminopeptidase 1 hr/30 “C

500 550 600 650 700750 800 850 900 950 1000

Leu-Trp-Met-Arg-Phe-Alo aminopeptidase 3 hr/30 ‘C

393

304 I

0 100 200 300 400 500 600 700 800 900 1000

Leu-Trp-Met-Arg-Phe-Alo ominopeptidase 1 hr/40 “C

‘;:;, ,11 /[j, ,

0 100 200 300 400 500 600 700 800 900 1000

Mixture of A-peptidase Digestion Products : 1 hr /30 ‘C , 3 hr/30 “C , 1 hr/40 %

Fig. 8. FD mass spectra of aliquots obtained from partial digestion of Leu-

Trp-Met-Arg-Phe-Arg in the same buffer as in Fig. 6 with aminopeptidase

M. a, 3O”C, 1 hr; b, 3O”C, 3 hr; c, 4O”C, 1 hr; d, mixture of equal volumes

of digests a, b, and c (35).

protein should be denatured and that it should stay soluble in the reaction buffers. Generally, the substrate is heated to 100°C for 2-3 min in order to denature it. In many cases, this treatment is not enough for denaturation and also the treated substrate becomes insoluble in the reaction buffer.


C

Fig. 9. Effects of detergents and alcohol on enzyme reactions. A and B: peptides (2 nmol) in 50 ,ul of 0.1 M pyridine, collidine-acetate, formate buffers containing various amounts of detergents or alcohols were digested with 5 ,og of enzymes for 16 hr at 37°C. The reaction mixture was analysed by an amino acid analyser for the liberated ammo acids. C and D: nitroanilide derivatives of N-terminal blocked peptides (200 nmol) in 500 ~1 of the volatile buffer containing various amounts of detergents or alcohols was digested with 5 pg of enzyme. Absorption at 380 nm was scanned wers’sus time at 20°C with a spectrophotometer. Time (T) required to reach a half height was read and values were expressed as proportions (%) of l/5 to those of the absence of detergents or alcohols. A: - octyl POE; --- SDS (0 SDS (20°C) in f and h) at 37°C for 16 hr. Underlined amino acids or peptides were analysed. a, carboxypeptidase A, Leu-Tip-&, pH 8.2; b, carboxypeptidase B, Leu- Tip-Met-&, pH 8.2; c, carboxypeptidase P, Leu-Tip-M>, pH 5.5; d, carboxypeptidase P, Leu-Trp-i&t, pH 2.5 ; e, trypsin, Leu-Trp-Met-Arg- &, pH 8.2, 37°C; f, same as e but 37°C and 2°C; g, Staphyloccocus aweus

protease V8, Gly-Glu-Gly-Phe-Leu-Gly-DPhe-Leu, pH 6.5; h, same as g but 37°C and 20°C. B: l methanol; 0 ethanol; A n-propanol at 37°C for 16 hr, substrates used were the same as in experiment A. a, carboxypeptidase A, pH 8.2; b, carboxypeptidase B, pH 8.2; c, carboxypeptidase P, pH 5.5; d, carboxypeptidase P, pH 2.5; e, trypsin pH 8.2; f, S. ourws protease V8, pH 6.5. C: l octyl POE; A Triton X-100; 0 SDS at 20°C pH 8.2. a, trypsin, beneoyl-Phe-Val-Arg-p-nitroanilide; b, submaxillaris protease, substrate is same as a; c, a-chymotrypsin, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; e, proteinase K, the substrate is the same as in c; f, pronase, the substrate is the same as in c. D: l methanol; 0 ethanol; A propanol at 2O”C, pH 8.2, substrates used were the same as in experiment C. a, trypsin; b, submaxillaris protease; c, a-chymotrypsin; d, subtilisin; e, proteinase K; f, pronase.

106 A. TSUGITA

Detergents are known to accelerate the denaturation and increase the solubility of protein. It was also observed that the addition of organic solvents such as alcohols causes similar effects and additionally they may change the hydrophobic protein conformations so that they are digestible by certain kinds of proteases (39). Based on this, we recently tested several protease activities in aqueous solution containing detergents or alcohol. Detergents: SDS, and lauryl glutamic acid (LG); neutral detergents: octyl polyoxyethylene (octyl POE) and Triton X-100; and alcohols: methanol, ethanol, and n-propanol were tested. Figure 9 summarizes these results (33). The data indicated the following: chemical reactions such as cyanogen bromide cleavage and Edman degradation reactions are stimulated by the addition of SDS, while a small amount of inhibition was observed upon the addition of Triton X-100 and octyl POE (data not shown). In contrast, as shown in Fig. 10, octyl POE shows little inhibitory effect on peptidase and protease activities and activation was even observed in a few cases. LG followed with a slightly higher inhibitory effect and SDS inhibited extensively. LG cannot dissociate and is insoluble at pH 2.5, therefore carboxypeptidase P activity was not tested with LG at this pH. Carboxypeptidase P in general has an optimal pH at 5.5, but it is often used at pH 2.5 because peptide bonds containing acidic amino acids are digestible only at this lower pH. Also, carboxypeptidase P at pH 2.5 is free from the endoprotease activity occasionally found at neutral pH. Inhibition by Triton X-100 was stronger than by octyl POE. Considerable inhibitory effects were seen after the addition of SDS. It is noticeable that even in the presence of SDS, carboxypeptidase B is almost fully active. The order of inhibition by alcohol correlates to the length of the carbon chain in the alcohols.

Since SDS is one of the most useful detergents known but at the same time exhibits the greatest inhibitory effect, a more detailed study was made of its effect.

Figure 10a demonstrates the effect on enzyme activity of varying enzyme concentrations at a constant volume (100 ~1) and a constant detergent concentration which alters for the individual enzymes. Except for carboxypeptidase B, an increase in the concentration of an enzyme pro- duces a relative increase in enzyme activity. This observation suggests that SDS concentration is not a crucial factor for enzyme inhibition but rather that the relative amounts of these enzymes to SDS is a more important factor.

In Fig. lob, both SDS concentration and the amount of enzyme were maintained at constant levels but the reaction volume was varied. The amount of SDS was increased by increasing the volume but the


0 El

10 20

h) l&l

1

2

100

50

I- O 10 20

P9 2

100

505u\

t w 0 100 /Jl

2

100 .

50 q 0 100 500

/I’

PI 3

100

50

I3 0 100 500

PI

fll 4

100

50 q 0 100 500

P’

Fig. 19. Effects of SDS on carboxypeptidase activities. The experimental

conditions were the same as described in Fig. 9 except for the following: a:

the enzyme concentration was changed from 0 to 25 pg/lOO ~1. 1, carboxy-

peptidase A; 2, carboxypeptidase B; 3, carboxypeptidase P (pH 5.5); 4, car-

boxypeptidase pH (2.5). The concentration of SDS was kept constant for

each of the enzymes through each set of experiments; 1% for carboxypepti-

dase A, 0.1% for carboxypeptidase B and P (pH 5.5) and 0.005% for car-

boxypeptidase P (pH 2.5). The final volume of the reaction mixture was

100 pl. b: volume of the reaction mixture was changed from 10 to 500 pl.

The concentration of SDS was kept constant for each set of experiments:

1 y0 for carboxypeptidase A, and O.l”/ f D or carboxypeptidases B and P (both

pH 5.5 and 2.5). The amount of enzymes was kept constant for each set of

experiments; 1, 50 pg for carboxypeptidase A; 2, 50 pg for carboxypeptidase

B; 3, 25 pg for carboxypeptidase P for pH 5.5 ; 4, 25 ,ug for carboxypeptidase

P pH 2.5. c: the volume of the reaction mixture has changed from 10 to

500 pl. The amount of SDS was constant (but the concentration was varied)

for each set of experiments: 1 mg for carboxypeptidase A, 0.1 mg for car-

boxypeptidase B and P (pH 5.5), and 5 ,cg for carboxypeptidase P (pH 2.5).

The amount of enzyme was kept constant for each set of experiments; 1,

50 pg of carboxypeptidase A; 2, 2.5 /Jg of carboxypeptidase B; 3, 10 pg of

carboxypeptidase P (pH 5.5); 4, carboxypeptidase P (pH 2.5).

amount of enzyme remained constant; the relative ratio of SDS/enzyme was increased with the increase of volume. This figure shows that as the

108 A. TSUGITA

Detergent (%)

Fig. 11. Effect of SDS on carboxypeptidase B activity on Leu-Trp-Met-

Arg. Concentration of SDS was changed from 0 to 1.0%. The substrate was

the same as experiment Fig. lb. Five micrograms of carboxypeptidase was

added in 100 ,ul of the reaction mixture. The solution was kept for 3 hr at

37°C. Liberated Arg (0) and Met (0) were determined with an amino acid

analyser.

relative ratio of SDS increased the enzyme activity became more inhibited. When the relative ratio of SDS/enzyme was kept constant the en-

zyme activity was constant regardless of variation ix-r the reaction volume. When the volume was increased, the SDS concentration decreased because a constant amount of SDS was in the reaction volume. Except for carboxypeptidase P at pH 2.5, the above prediction was experimentally proved as shown in Fig. 10~. It is known that carboxypeptidase P is unstable at pHs lower than 2.0. Carboxypeptidase P activity at pH 2.5 was peculiarly observed to increase with decreased SDS concentration. This may be the result of the enzyme being unstable at lower pHs.

As shown in Fig. 11, SDS inhibits carboxypeptidase A but hardly affects carboxypeptidase B activity. In practice, commercial preparations of carboxypeptidase B are almost always accompanied by carboxypeptidase A contamination. We may use SDS inhibition to obtain pure carboxypeptidase B activity. In Fig. 9, the substrate Leu-Trp-Met-Arg was used to test this proposition. In the absence of SDS, a carboxypeptidase B preparation liberated not only the theoretical amount of Arg but also 75% of Met. When SDS ( more than O.lo/o) was added, the liberation of Met dramatically dropped but the liberation of Arg was maintained. Arg liberation was not inhibited, even with the addition of up to 1% SDS. This and other experiments on proteins show the practical use of SDS for C-terminal determination by carboxypeptidase B digestion without carboxypeptidase A activity (57).

DEVRL.OPMRNTS IN PROTEIN MICROSEQUENCING 109

It should be noted that these observations are not limited to carboxypeptidases but also extend to endoproteases such as trypsin and Staphylococcus aweus V8 protease. A similar type of experiment to that in Fig. 10a was made with these two enzymes, and both trypsin in 0.01% SDS and V8 protease in 0.05% SDS resulted in comparable curves to that of carboxypeptidase A.

Using the above data one may denature a protein in a concentrated detergent or alcohol solution, then dilute the solution to an adequate concentration and digest the protein with carboxypeptidases (or proteases). In many cases thus treated proteins were easily dissolved, sufficiently denaturated and readily digested with carboxypeptidases (6, 12-15).

v. MONITORING OF AMINO GROUPS AT THE BINDING SITES OF PRO-

TEINS (54)

Most chemical modifications of proteins potentially cause alterations in conformation. Afhnity labeling, especially photoaffinity labeling is one of the techniques used to minimize these alterations. However, the efficiency of the photoaffinity labeling is not always lOOo/o, which complicates peptide mapping procedures (53). Recently we established a chemical modification method to monitor the NH, group(s) on the surface of the DNA binding protein in both the presence and absence of DNA. The reagent used is ethyl acetimidate which reacts with NH, groups of proteins under mild conditions and introduces imino groups (54). The introduction of the imino groups instead of the NH, groups does not change the surface charges of the proteins and consequently minimizes the alteration of the protein conformation (55).

The protein, Pfl phage single stranded DNA binding protein (53), in the nucleoprotein complex form was reacted with a 10 times molar excess of ethyl acetimidate in 50 rnM NaHCO, (pH 8.4) for 2 hr at 20°C. After the reaction, the excess reagent was removed by dialysis and the DNA was removed by centrifugation. An aliquot of protein was tested for DNA binding activity. When the reaction was carried out under adequate conditions keeping the DNA-protein complex, the binding site of the protein was covered with DNA which did not react with the reagent, and the modified protein kept its binding activity. The modified protein was then labeled again by addition of W-acetimidate reagent. An aliquot of the second modified protein after freeing the excess reagent was tested to confirm that the binding activity was lost. Peptide mapping was carried out after digestion of the modified protein with chymotrypsin, which located the radioactively labeled peptide on the sequence. The experiment

110 A. TSUGITA

was also performed in the reverse order, i.e., the radioactive reagent was added to the complex and the unlabeled reagent added after removal of DNA.

Modification of the NH,-groups in the native nucleoprotein complex showed that 7 out of the 8 lysines present and the N-terminus were accessible to the reagent and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. A specific lysine was identified by peptide mapping as the major protected residue. Modification of this residue abolished DNA binding activity.

The DNA in the nucleoprotein complex was replaced by a synthetic octanucleotide. A similar experiment was performed and peptide mapping again revealed a specific lysine was radioactively labeled. This type of experiment aims to monitor the area in the molecule protected by other molecules, which can be either DNA or protein. The active conformation being undisturbed after the first modification is one of the most important advantages of this method and it may be achieved by using a special reagent such as ethyl acetimidate for the amino group which does not introduce a large molecule nor cause ionic disturbance.

SUMMARY

The author has described here several methods which have been developed in his laboratory. These methods are explained in the following order: [I] gel electrophoresis; 1) extraction from the conventional polyacrylamide gel, 2) a new polyacrylamide gel electrophoresis system for small peptides. [II] Amino acid composition (rapid and micro methods for hydrolysis of proteins) ; 1) hydrolysis with a mixture of trifluoroacetic acid and hydrochloric acid, 2) vapour hydrolysis, 3) tryptophan microanalysis. [III] Amino-terminal sequencing; 1) a sensitive detection method of Edman degradation, 2) N-terminal specific labeling method. [IV] Carboxy-terminal sequencing by carboxypeptidase digestion; 1) field desorption mass spectrometry, 2) the use of detergents or alcohols. M Monitoring of amino groups at the DNA binding site of protein.

As mentioned in the beginning, techniques in the protein field tend to be behind comparable techniques in the DNA field. In addition, the fact that the most important biological molecules presently studied are made in such small amounts in the cell has demanded the development of techniques using precisely and quickly only micro amounts of protein. Such developments of protein chemistry techniques for purification, composition, sequencing, and chemical modification are very much needed


in the cell biology field and close collaboration is required between protein chemistry, biophyiscs, and modem biology in order to develop the above methodology.

Acknowle&ments I would like to thank the following people for their help, either for their work in the new developments covered in this review and/or for their advice and discussions: Dr. I. Arai, Dr. M. Kamo, Mrs. H. W. Mewes, T. Ataka, F. Vilbois, and C. S. Jone in my laboratory.

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Received for publication April 3, 1987.

developments in protein microsequencing

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