[19]
E N Z Y M A T I C A N D C H E M I C A L D I G E S T I O N OF PROTEINS
361
[ 19] E n z y m a t i c a n d C h e m i c a l Digestion of P r o t e i n s for M a s s S p e c t r o m e t r y
By TERRY D. LEE and JOaN E. SHIVELY Introduction The standard approach for obtaining partial or complete structures of proteins without resorting to cloning cDNAs is to digest the purified protein with one or two selected endoproteases or by chemical cleavage methods, then to fractionate the resulting peptides by reversed-phase high-performance liquid chromatography (HPLC), and to sequence the resulting peptides. In the past, peptides have been primarily sequenced by automated Edman chemistry, which currently has a sensitivity in the mid to low picomole range. If possible, structures were confirmed by amino acid compositional analysis which revealed the amount of sample present and the relative molar ratios of each amino acid. With the advent of mass spectrometric techniques such as fast atom bombardment mass spectrometry (FAB-MS), it has been possible to obtain molecular weights for the majority of peptides analyzed, even at the low picomole range. For laboratories with access to MS/MS approaches (see [24] and [25] in this volume), it is often possible to obtain complete structures by mass spectrometric methods alone. Historically, nanomole amounts have been required for sequencing by mass spectrometry. However, as instrumentation and methods improve, more and more problems can be solved with sample amounts in the 100 pmol range. With these objectives in mind, we present here methods for obtaining peptide maps at the 100 pmol level on widely available standard proteins (horse cytochrome c and myoglobin). In order to simplify the protocol, standards were chosen that do not require reduction and S-alkylation prior to digestion. However, it should be noted that many proteins are proteaseresistant unless this step is performed first. For each method described, a peptide map is shown (see Fig. 1), from which selected peaks were analyzed by FAB-MS to demonstrate that the method is compatible with standard FAB-MS procedures (representative spectra are given later in Fig. 2). For convenience, the sequences for horse heart cytochrome c and horse heart myoglobin are given in Fig. 3 and Fig. 4 (see later), respectively; those peptides that were characterized are indicated. It should be noted that this is not an exhaustive treatment of all available methods, but is a compilation of the more commonly encountered approaches. While it METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
362
PEPTIDES AND PROTEINS
[19]
is undoubtedly easier to obtain results at > I nmol level, it was thought that the majority of researchers already obtain good results at these higher levels, but often encounter difficulties when working at the 100 pmol level or below. Furthermore, it is good practice to validate a method on a readily available standard protein before attempting the experiment on a precious unknown. Thus, these protocols will serve as a starting point to confirm that the method is working, and the hardware involved (HPLC, mass spectrometer, sequencer, amino acid analyzer, etc.) is functioning properly. No attempt is made to optimize or discuss the HPLC peptide mapping procedures (this topic is also covered in [21] in this volume).
Materials and Sources Horse heart cytochrome c, Sigma (St. Louis, MO) Horse muscle (apo)myoglobin, Sigma Bovine trypsin (TPCK-treated), Sigma Chymotrypsin, Sigma Staphylococcus aureus protease V8 (endoproteinase Glu-C), Boehringer Mannheim (Indianapolis, IN) Endoproteinase Lys-C (Lysobacter enzymogenes), Boehringer Mannheim Endoproteinase Lys-C (Achromobacter lyticus), Wako Pure Chemicals (Dallas, TX; Osaka, Japan) Endoproteinase Arg-C (mouse submaxillary gland), Boehringer Mannheim Endoproteinase Asp-N (Pseudomonas fragi), Boehringer Mannheim Pepsin, Sigma Thermolysin, Sigma N-Chlorosuccinimide, Sigma Cyanogen bromide, Sigma Ammonium bicarbonate, Mallinckrodt (St. Louis, MO) Trifluoroacetic acid (sequencer-grade), Pierce (Rockford, IL) Acetonitrile (HPLC-grade), Baker (Phillipsburg, N J) Dimethyl sulfoxide (DMSO), Baker Dithiothreitol (DTT)/dithioerythritol (DTE), Calbiochem (San Diego, CA) Disodium phosphate, Sigma Alternative enzyme sources include Calbiochem, Worthington, and Pierce. Except as noted, the enzymes were used without further purification. Solutions and Preparation Ammonium bicarbonate, 1.0 M (pH 8.5): 3.95 g N H 4 H C O 3 in 500 ml water
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
363
Ammonium bicarbonate, 0.1 M (pH 8.5): dilute above 1/10 in water Trifluoroacetic acid, 10% (TFA): add 1 ml of TFA to 9 ml of water Tris-HCl, 0.1 M (pH 9.0): add 12.1 g of Tris base to 900 ml of water, titrate to pH 9.0 with 12 M HCI, and dilute to I liter Sodium phosphate, 0.10 M, EDTA, 4 mM (pH 7.4): 1.42 g of disodium phosphate and 0.15 g sodium EDTA in 90 ml water, adjust pH to 7.4 with 10% phosphoric acid, and dilute to 100 ml HPLC, solvent A: 1 ml TFA in 999 ml water HPLC, solvent B: 1 ml TFA, 99 ml water, 900 ml acetonitrile Water was obtained from a MilliQ system. All solutions were stored at 4 ° unless otherwise noted. Mass Spectrometry All mass spectrometric analyses are performed on a JEOL HX100HF double-focusing magnetic sector mass spectrometer having modified Nier-Johnson geometry. The instrument is operated at 5 kV accelerating voltage and a nominal resolution setting of 3000. Sample ionization is accomplished using a xenon atom beam having 6 keV translational energy. Unless otherwise noted, dried, HPLC-purified samples in polypropylene tubes are taken up in 2/.d of DMSO and applied to a 1.5 mm x 6 mm stainless steel sample stage which had previously been covered with ca. 2/zl of the DTT : DTE (5 : 1) matrix. Spectra are recorded over the mass range of 300 of 3500 m/z and mass assignments made using a JEOL DA5000 data system. Unless otherwise noted, mass values reported are for the monoisotopic protonated molecular ion. Endoproteinase Lys-C ( Achromobacter lyticus ). The enzyme (3.3/.~g) is dissolved in 1 ml water, divided into 100-/zl aliquots, and stored at - 20°. The enzyme is diluted (5/zl in 1 ml) with 0.1 M Tris buffer just before use. The sample, cytochrome c (1.2/xg, 100 pmol) in 20/zl of 0.1 M Tris buffer (pH 9.0), is mixed with 100/zl of diluted enzyme (1 : 200 tool/tool; 0.00165/zg), and digested for 6 hr at 30°. The sample is neutralized with 10/.d of 10% TFA and chromatographed on a Vydac Cls column (2.1 x 150 ram) on a Beckman System Gold Chromatograph using a linear gradient from 98% A to 70% B over 60 min with a flow rate of 0.2 ml/min (214 nm detection, 0.08 AUFS) (Fig. 1). Peaks are manually collected in 1.5-ml polypropylene tubes and stored at - 20° prior to mass spectrometric analysis (Table I) (Fig. 2). This enzyme is more aggressive than the enzyme from Lysobacter. It can be used at an enzyme to substrate ratio of 1/200 to 1/400, and tolerates moderate amounts of urea (1-4 M) and SDS (0.1%). 1 1 K. Morihara, T. Oka, H. Tsuzuki, Y. Tochino, and T. Kanaya, Biochem. Biophys. Res. Commun. 92, 396 (1980).
364
PEPTIDES AND PROTEINS
Lys C (Achromobacter)
[19]
I Endo Asp-N Myoglobin
1'5 ' 2'5 Lys C (Lysobacter)
3'5 rnin
'
10
....
jAN-2
I
I
I
20
I
30 min
AH 1'5
'
2'S
'
3'5 min
I
I0
I
I
20
I
50 rain AC-I
Endo Argc
My~lol~n
1'5
'
2'5
'
Chymotrypsin Cytochrome c
!
:35 min
i
!
|
50 min i
30
Thermolysin Myoglobln
Th-1
i
C-2
I
I
I
20
I
30 min
;;'°PScCr°me c
I
20
I
I
30 rain
I
I
I
I
10 30 Cyanogen Bromide Cytochrome c
40
I
50 min i i
!
!
60
80
!
100 min
FIG. 1. HPLC separations of enzyme and chemical digests of cytochrome c and myoglobin. Labeled peaks were analyzed by FAB mass spectrometry (see Tables I and II). The sequence of the peptides is shown in Figs. 3 and 4.
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
365
TABLE I EXPERIMENTAL AND CALCULATEDMASS VALUES FOR PROTONATED MOLECULAR IONS IN MASS SPECTRA OF SELECTED DEGRADATION PRODUCTS FOR HORSE HEART CYTOCHROME C MH + HPLC
Enzyme
fraction
Observed m/z
Calculated m/z
Endoproteinase Lys-C
LA-1 LA-2a LA-2b LL-I LL-2 T- 1 T-2a T-2b C-1 C-2 P-1 P-2 CN- 1 CN-2
604.3 1350.8 1478.8 779.5 1296.7 1470.7 1350.8 1478.8 803.4 1162.5 966.7 1162.6 1763.2 2780.0
604.4 1350.7 1478.8 779.5 1296.8 1470.7 1350.7 1478.8 803.5 1162.6 966.6 1162.6 1762.9 2779.6
(Achromobacter) Endoproteinase Lys-C
(Lysobacter) Trypsin
Chymotrypsin Pepsin Cyanogen bromide
Endoproteinase Lys-C (Lysobacter enzymogenes). The enzyme (3 U, 100/zg) is mixed with 200/zl of water. The enzyme is active for at least 1 month when stored at 4 ° and is diluted 1 : 100 with water just before use. The sample, cytochrome c (1.4/xg, 120 pmol) in 50/zl of water, is mixed with 2-3/.d of 1.0 M ammonium bicarbonate buffer (pH 8.5) and 5/zl of diluted enzyme solution (0.05/zg; 1 : 30, w/w) in a 1.5-ml polypropylene tube, and digested for 24 hr at 37 °. The sample is directly chromatographed on a Vydac C4 column (2.1 x 250 mm) on a Brownlee Microgradient System using a linear gradient from 98% buffer A to 100% buffer B over 60 min with a flow rate of 0.15 ml/min (214 nm detection, 0.16 AUFS) (Fig. 1). Peaks are manually collected in 1.5-ml polypropylene tubes stored at 4 ° prior to analysis by mass spectrometry (Table I). Standard-grade enzyme is used rather than the more expensive sequencing-grade enzyme. This enzyme cleaves on the C-terminal side of lysine residues. Trypsin. Trypsin is further purified by reversed-phase HPLC according to Titani et al. 2 Trypsin (250/xl; 1/zg//~l) is purified on a Brownlee Aquapore C4 column (4.6 x 30 mm) on a Beckman System Gold HPLC using a linear gradient from 100% A to 100% B over 60 min with a flow rate of 2 K. Titani, T. Saagawa, K. Resing, and K. A. Walsh, Anal. Biochem. 123, 408 (1982).
366
PEPTIDES AND PROTEINS
100
[19]
!A 385
LA-1 604
(.) t-
'1o t:~ 50G)
.>_. (D
n,-
425
503
l.lk
| I
.
.
.
.
I
400
.
.
.
.
500
.
.
.
.
I
600
700
.
.
.
-
.
.
"
.
900
800
100
m/z 100
B
LA-2a 1351
8t "1o t50a)
._> LA-2b 1479
(9
cC
i~~'~ta-'='''
J~ ~"1
"
"
I
500
.
.
.
.
.
.
.
.
"~' ~ ' =
.
I
.
.
,,,..~= jld .
.
.
.
.
1000 m/z
a.J....L,.l,._L,id,~.., h , "
"
I
1500
.
.
.
.
.
.
.
.
.
.
2000
~'~
FIG. 2. Positive ion FAB mass spectra of(A) HPLC fraction LA-1 and (B) HPLC fraction LA-2. Spectra are representative of those obtained from other samples.
0.5 ml/min (214 or 280 nm detection). The major peak eluting at 43% B is manually collected into a 1.5-ml polypropylene vial, dried in a vacuum centrifuge, redissolved in 1 ml of water, and stored frozen in 20-/xl aliquots. The amount collected is verified by amino acid analysis (20/zl gave 2.5 /zg). The enzyme is diluted (20/xl in 1 ml) of 0.1 M ammonium bicarbonate just before use. The sample, cytochrome c (1.2/xg, 100 pmol) in 20/zl of 0.1 M ammonium bicarbonate buffer (pH 8.5) in a 1.5-ml polypropylene tube, is mixed with 100/~1 of HPLC-purified, diluted trypsin (1 : 50 w/w; 0.025/zg), and
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
367
digested for 18 hr at 37 °. The sample is neutralized with 10/~1 of 10% TFA, and chromatographed on a Vydac Cl8 column (2.1 × 150 mm) on a Beckman System Gold using a linear gradient from 98% A to 7Q% B over 60 min with a flow rate of 0.2 ml/min (214 nm detection, 0.08 AUFS) (Fig. I). Peaks are manually collected in 1.5-ml polypropylene tubes and stored at - 2 0 ° prior to FAB-MS analysis (Table I). Trypsin cleaves on the C-terminal side of lysine and arginine residues, but not at Lys-Pro, and occasionally not at Arg-Pro. The trypsin must be purified to avoid spurious cleavages due to contamination with chymotrypsin. Trypsin can tolerate small amounts of urea (1-2 M), but is inhibited by sodium dodecyl sulfate (SDS) and guanidine-HC1. If the sample is treated with urea, the urea must be freshly deionized. Trypsin may also tolerate small amounts of propanol or acetonitrile (10-20%, see Ref. 3). Chymotrypsin. Chymotrypsin is HPLC-purified as described above for trypsin. The final concentration is 0.05 ttg//~l. Although Titani and coworkers 2 included calcium chloride in the HPLC buffer to maintain the activity of chymotrypsin, we have found it unnecessary. The sample, cytochrome c (1.2/.~g, 100 pmol) in 25/.d of 0.1 M ammonium bicarbonate buffer (pH 8.5) in a 1-ml polypropylene tube, is mixed with 3/~1 (0.0075/zg) of HPLC-purified chymotrypsin (1 : 160, w/w), and digested for 18 hr at 37 °. The sample is neutralized with 3 ~1 of 50% acetic acid, diluted to 100 ~1 with 0.1% TFA, and chromatographed on a Vydac Cls column (2.1 x 250 mm) on a Brownlee Microgradient System using a linear gradient from 98% A to 100% B over 60 min with a flow rate of 0.15 ml/min (214 nm detection, 0.08 AUFS) (Fig. 1). Peaks are manually collected in 0.5-ml polypropylene tubes stored at - 2 0 ° prior to analysis by mass spectrometry (Table I). Chymotrypsin generally cleaves on the C-terminal side of hydrophobic and aromatic residues, such as, leucine, phenylalanine, tyrosine, tryptophan, and, occasionally, at histidine. HPLC purification of chymotrypsin removes contaminating trypsin. The sequence of horse heart cytochrome c is shown in Fig. 3. Endoproteinase Asp-N. The enzyme (2.0/.~g) is dissolved in 50/zl of water and stored at - 2 0 °. The enzyme remains active when tested over a period of 1 to 2 months. Horse muscle myoglobin is prepared at a concentration of 0.7/.~g//~l in 5% acetic acid and stored at 4°. The sample, myoglobin (1.7/zg, 110 pmol) in 2.5/zl of 5% acetic acid and 40/~1 of 0.05 M sodium phosphate buffer (pH 8.0) in a 1-ml polypropylene tube, is mixed with 1.0 ~1 of enzyme (1 : 50 w/w; 0.04 ~g), and digested for 18 hr at 20°. The sample is neutralized K. Gjesing Welinder, Anal. Biochem. 174, 54 (1988).
368
PEPTIDES AND PROTEINS
1 tGDVEKGKK I C-2/P-2
GI
10 20 30 I FVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNK 4 ) (1162)
60 T W K E E T L M E Y L E N P KI K Y I
70
80
PGTKMI CN-I
FAGI
CN-2 LL-I
40
I (779)
50 I
1,5-2 (1296)
90 KKKTEREDL
(1763) l
[19]
IAY
100 LKKATNE
4
(2780)
I
'1
T-2b/LA-2b I T-2a/LA-2a
(1478) (1350} I I P-1 i C-1
(966)
I I
(803)
FIG. 3. Sequence of horse heart cytochrome c and location of the fragments analyzed by FAB mass spectrometry. The N-terminal glycine (*G) is acetylated.
with 10/xl of 10% TFA and chromatographed on a Vydac C~s column (2.1 x 100 mm) on a Beckman System Gold Chromatograph using a linear gradient from 95% A to 65% B over 65 min with a flow rate of 0.2 ml/min (214 nm detection, 0.16 AUFS) (Fig. 1). Peaks are manually collected in 1.5-ml polypropylene tubes and stored at - 20° prior to analysis by mass spectrometry (Table II). This enzyme cleaves on the N-terminal side of aspartic acid, and sometimes glutamic acid residues. In performic acid oxidized samples, it will also cleave at cysteic acid residues. The enzyme can tolerate small amounts of SDS (0.1%). Endoproteinase Glu-C (Staphylococcus au?eus). The enzyme (50/zg) is dissolved in 50/xl of water and stored at 4°. The sample, myoglobin (1.7/zg, 100 pmol) in 2.5/zl of 5% acetic acid in a 0.5-ml polypropylene tube, is mixed with 12.5/zl of 0.1 M sodium phosphate, 4 m M EDTA buffer (pH 7.4), 4/zl of water, and 6/zl of the enzyme solution (0.06 /zg) diluted 1:100 with water (1:30 w/w), and digested for 16 hr at 37°. The sample is acidified with 0.5/zl of 50% acetic acid and diluted to 100/~1 with 0.1% aqueous TFA prior to fractionation over a Vydac Cls column (2.1 x 250 ram) on a Brownlee Microgradient System. Digestion products are eluted using a linear gradient from 98% buffer A to 100% buffer B over 60 min with a flow rate of 0.15 ml/min (214 nm detection) (Fig. 1). Peaks are manually collected into 0.5-ml polypropylene tubes and stored at - 20° prior to mass spectrometric analysis (Table II). In the conditions described above, the enzyme cleaves on the C-terminal side of glutamic and aspartic acid residues. In 0.5 M ammonium acetate (pH 4.0), the enzyme is reported to cleave exclusively after glutamic acid. 4 4 j. Houmard and G. R. Drapeau, Proc. Natl. Acad. Sci. U.S.A. 69, 3506 (1972).
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
369
TABLE II EXPERIMENTAL AND CALCULATED MASS VALUES FOR PROTONATED MOLECULAR'IoNs IN MASS SPECTRA OF SELECTED DEGRADATION PRODUCTS FOR HORSE HEART MYOGLORIN MH + Enzyme Endoproteinase Asp-N
Staphylococcus aureus protease Endoproteinase Arg-C Thermolysin
HPLC fraction
Observed m/z
Calculated m/z
AN-1 AN-2 S-I S-2 AC-I Th-l Th-2
1439.6 1858.0 822.6 664.4 3405.6 a 1345.4 1420.4
1439.7 1857.9 822.5 664.3 3405.7 ~ 1345.6 1420.8
Average mass. Other values are monoisotopic masses.
With myoglobin, however, no reaction occurs under these conditions. The enzyme can tolerate small amounts of SDS (0.05-0. I%). Endoproteinase Arg-C (Mouse Submaxillary Gland). The enzyme (100 units, 265/~g) is mixed with 100/~l of water. The enzyme is active for 2 to 3 months when stored at - 2 0 °. The enzyme solution is further diluted 1 : 250 just before use. The sample, apomyoglobin (1.7/.~g, 100 pmol) in 2/~l of water in a 0.5-ml polypropylene tube, is mixed with 100/~l of 0.1 M ammonium bicarbonate buffer (pH 8.0) and 3/~l of the diluted enzyme solution (0.032 /~g; 1:50, w/w), and digested for 24 hr at 37°. The sample is directly chromatographed on a Vydac C18 column (2.1 x 250 mm) on a Beckman System Gold. Digestion products are eluted using a linear gradient from 95% buffer A to 60% buffer B over 60 min with a flow rate of 0.25 ml/min (214 nm detection, 0.16 AUFS) (Fig. 1). Peaks are manually collected into 1.5-ml polypropylene tubes and stored at - 2 0 ° prior to analysis by mass spectrometry (Table II). The enzyme cleaves at the C-terminal side of arginine residues. 5 In this example, some cleavage at lysine is also observed. Thermolysin. Thermolysin is prepared at a concentration of 1.0/.~g//.d in water and stored at - 20°. The enzyme remains active for 1 to 2 months. The enzyme is diluted 1 : 20 in water just prior to use. The sample, myoglobin (1.7/~g, 110 pmol) in 2.5/.d of 5% acetic acid and 40 /~l of 0.1 M ammonium bicarbonate buffer (pH 8.5) in a 1-ml 5 M. Levy, L. Fishman, and I. Schenkein, this series, Vol. 19, p. 672.
370
PEPTIDES AND PROTEINS
[19]
G L S D G E W Q Q V L N V W G K V E A D I A G H G Q E V L I R L F T G H P E T L E K F D K F K H L K T E A E M I i AC-1 (3405) I
Th-2 I
I
(1345) AN-2
(1345}
I
Th-2
I
(1420)
I
60 70 80 90 i00 K A S E D L K K H G T V V L T A L G G I L K K K G H H E A E L K P L A Q S H A T K H K I P I K Y L E F I S D A 120 I I HVLH
SKHP
130 G N F G A D A Q G A M T K A L E L F
140 RND I S-I
150 KELGFQG
I AAKY I
ii0
AN-I
I (664) I
(1439) I
S-2
(822)
FIG. 4. Sequence of horse muscle (apo)myoglobin and location of the fragments analyzed by FAB mass spectrometry.
polypropylene tube, is mixed with 2.0/.d of 1 : 20 diluted enzyme (1 : 20, w/w; 0.08 mg), and digested for 20 min at 40°. The sample is neutralized with 10/zl of 10% TFA and chromatographed on a Vydac C18 column (2.1 x 100 mm) on a Beckman System Gold using a linear gradient from 95% A to 65% B over 65 min with a flow rate of 0.2 ml/min (214 nm detection, 0.16 AUFS) (Fig. 1). Peaks are manually collected into 1.5-ml polypropylene tubes and stored at - 20° prior to mass spectrometric analysis (Table II). Thermolysin cleaves at the N-terminal side of leucine, isoleucine, valine, phenylalanine, methionine, and alanine, often resulting in the formation of very small peptides. Thus, digestion conditions must be adjusted accordingly. The enzyme can be used on proteins resistant to proteases or for cleavage between disulfide bonds. It can be seen that digestion is incomplete for this example; however, longer digestion conditions may result in degradation of the peptides to di- to tetrapeptides. Appropriate digestion times may be ascertained by removing aliquots at hourly intervals, followed by analytical HPLC analysis. The sequence of horse muscle myoglobin is given in Fig. 4. Pepsin. Pepsin (636/zg) is dissolved in 20 ml of 1 mM ammonium acetate buffer (pH 7). The enzyme is made fresh each time before use. The sample, cytochrome c (1.2/xg, 100 pmol) in 95/zl of 0.01 M HC1 (pH 2.0), is mixed with 5/xl of the enzyme solution (1:7.5, w/w; 0.16/~g by amino acid analysis), and digested for 4 hr at 37°. The sample is directly chromatographed on a Vydac C18 column (2.1 x 150 ram) on a Beckman System Gold using a linear gradient from 98% A to 70% B over 60 rain with a flow rate of 0.2 ml/min (detection at 214 nm, 0.16 AUFS) (Fig. 1). Peaks are manually collected in 1.5-ml polypropylene tubes and stored at - 2 0 ° prior to analysis by mass spectrometry (Table I). Samples are dis-
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
371
solved in 2/~1 of 1% TFA for mass spectrometric analysis and scanned over the mass range of 700 to 3000. Pepsin cleaves at the C-terminal side of phenylalanine, methionine, leucine, and tryptophan residues adjacent to hydrophobic residues. The digestion of cytochrome c is incomplete, likely due to an impurity as reported by Yuppy and Paleus. 6 Pepsin has maximal activity at pH 2, but may autodigest when stored for extended periods at pH <4. Cyanogen Bromide. Cyanogen bromide (CNBr) (2 mg) is dissolved in 100/zl of 70% TFA and freshly made before each use. CAUTION"cyanogen bromide is extremely toxic and should be handled with gloves in a wellventilated fumehood. Since extremely small amounts are used here, the samples may be later dried in a vacuum centrifuge equipped with a dry ice trap. Carefully clean the trap following this procedure. Do not follow this procedure on scaled-up runs. See Vol. XI in this series for further details. 7 The sample, cytochrome c (1.2/zg, 100 pmol) in 5/xl of 70% TFA, is mixed with 100/zl of cyanogen bromide (2 mg), and digested for 2 hr at 25 °. The sample is dried on a vacuum centrifuge equipped with a dry ice trap, redissolved in 100/.d of 70% TFA, and chromatographed on a Vydac C~8 column (2.1 x 250 mm) on a Beckman System Gold Chromatograph using a linear gradient from 98% A to 60% B over 120 min with a flow rate of 0.25 ml/min (214 nm detection, 0.16 AUFS). The chromatograph is shown in Fig. 1. Peaks are manually collected in 1.5-ml polypropylene tubes and stored at - 2 0 ° prior to mass spectral analysis (Table I). CNBr cleaves at the C-terminal side of methionine residues, converting the methionines to a mixture of homoserine and homoserine lactone. Acidic conditions favor the lactone, while basic conditions favor the free acid. If desired, the interconversion can be affected prior to, or on the stage, during FAB-MS analysis. Some degradation of tyrosine and tryptophan residues may be observed. If the reaction is performed in formic acid, one may also observe reduction of disulfide bonds, a If the reaction is performed with heptafluorobutyric acid, some cleavage at tryptophan may occur. 9 CNBr fragments are often large and hydrophobic, and, therefore, tend to aggregate, making them difficult to separate by reversedphase HPLC. It is suggested that they be dissolved in a strongly denaturing solvent such as hexafluoroacetone trihydrate (HFA), 70% TFA, or 6 M
6 H. Yuppy and S. Paleus, Acta Chim. Scand. 9, 353 (1955). 7 E. Gross, this series, Vol. 11, p. 238. a S. Villa, G. De Fazio, and U. Canosi, Anal. Biochem. 177, 161 (1989). 9 j. Ozols and C. Gerhard, J. Biol. Chem. 252, 5986 (1977).
372
PEPTIDES AND PROTEINS
[19]
guanidine-HC1 before injection onto the reversed-phase column. Even under these circumstances, the peaks may be broad and obtained in low yields. In this example, better results are obtained with 70% TFA compared to 90% formic acid. N-Chlorosuccinimide. N-Chlorosuccinimide (NCS) (13.3 mg) is dissolved in 10 ml of 0.3 M dimethylformamide (DMF). The solution is freshly made before each use. The sample, cytochrome c (1.2 /~g, 100 pmol) in 5 txl of water, is mixed with 10/zl of 50% acetic acid, 5/zl of 0.01 M NCS in DMF, and digested for 2 hr at 25 °. The reaction is terminated with 4/xl of 0.08 M N-acetyl-L-methionine, and directly chromatographed on a Vydac C 4 column (2.1 × 250 mm) on a Beckman System Gold using a linear gradient from 90% A to 100% B over 60 min with a flow rate of 0.25 ml/min (214 nm detection, 0.04 AUFS). Peaks are collected manually in 1.5-ml polypropylene tubes and stored at - 20° prior to microsequence analysis. The chromatogram is not shown. Peaks are concentrated in a vacuum centrifuge to an approximate volume of 2-5 /.d, and subjected to microsequence analysis. The sequence obtained is KEETLMEYLENPKKYIPGTKMIFAG . . . . The fragment corresponds to residues 60-104 of cytochrome c. The N-terminal yield is 10 pmol, corresponding to an overall yield of 10% (losses may occur during the cleavage reaction, HPLC, and sequence analysis). NCS cleaves at the C-terminal side of tryptophan residues. 1° The reagent may also oxidize methionine to methionine sulfone, and may destroy some tyrosine residues. In this example, neither methionine nor tyrosine is affected. Other reagents which have been used to cause oxidative cleavage at tryptophan are CNBr/formic acid/HFBA 9 and iodosobenzoic acid. H Due to the rare occurrence of tryptophans in proteins, the expected fragments are very large, usually beyond the mass range of most instruments. The same arguments about solubility and low yields which apply to CNBr fragments also apply here. Due to the large expected fragment sizes in this example, no attempt is made to perform FAB-MS analysis.
Additional Comments
Each of these examples included an enzyme/buffer or reagent/solvent control (not shown). The control allows the subtraction of spurious peaks (should they occur), or an estimation of autodigestion in the case of 10 y. Shechter, A. Patchornik, and Y. Burstein, Biochemistry 15, 5071 (1976). II W. C. Mahoney, P. K. Smith, and M. A. Hermodson, Biochemistry 20, 443 (1981).
[19]
ENZYMATIC AND CHEMICAL DIGESTION OF PROTEINS
373
proteolysis. The conditions outlined here should only be used as a starting point. If volumes and concentrations are varied, one should first verify the appropriateness of the conditions on standards. Many proteins are protease-resistant, and will require prior reduction and S-alkylation (or at least denaturation). Unfortunately, this step usually requires desalting or repurification of the protein before proceeding to enzymatic digestion. The sample may be S-alkylated with iodoacetic acid ~2or 4-vinylpyridine. ~3A general protocol for the latter is given below. A reversed-phase HPLC method for desalting and repurifying the S-alkylated protein is given by Pan et al. ~2Another approach is to denature the protein in formic acid and break the disulfide bonds by performic acid oxidation. TMThis approach will also oxidize methionine and tryptophan residues, thus preventing cleavage by CNBr, but may be useful for Asp-N cleavage at cysteic acid residues. A representative protocol for perfot~mic acid oxidation is given below. Finally, in order to locate disulfide bonds, it will be necessary to maintain them intact through the digestion process. A separate chapter in this volume deals with this problem. Reduction/S-alkylation. Dissolve the sample in 100/zl of 6 M guanidineHCI, 0.1 M Tris-HCl (pH 8.0), and 10 mM EDTA. Flush the tube (serumcapped Reacti-Vial) with argon, add 1/~I of 100 mg/ml DTT, and reduce for 60 min at 30° to 50°. Add 1/xl of 4-vinylpyridine and alkylate for 30 to 60 min at 30° to 50°. Desalt the sample by reversed-phase HPLC.12 Performic Acid Oxidation. Dissolve the sample in 100 ~1 of 90 to 98% formic acid, chill to 0°, and add 1-10/zl of freshly prepared performic acid (add 250/~1 of 30% hydrogen peroxide to 4.75 ml of 90 to 98% formic acid for 2 hr at 25° and chill to 0°). Allow the reaction to proceed for 5 to 30 min and dry on a vacuum centrifuge. Redissolve the sample in an appropriate solvent such as 70% TFA or 100% HFA. This chapter presents methods which have been documented in our laboratory. Obviously, it is not comprehensive. Excellent protocols are available for other proteases, and for specific cleavage at aspartyl-proline peptide bonds, cysteine peptide bonds, etc. It is hoped that this will provide a starting point for newcomers to the field, or for researchers who have previous experience with digesting nanomole amounts of protein, but need a protocol for starting with picomole amounts of protein. Most importantly, the protocols are designed with further analysis by mass spectrometry in mind. 12 Y.-C. Pan, J. Wideman, R. Blacher, M. Chang, and S. Stein, J. Chromatogr. 297, 13 (1984). 13 A. S. Inglis, this series, Vol. 91, p. 26. 14 C. H. W. Hirs, this series, Vol. 11, p. 197.
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Acknowledgments The authors express their sincere thanks for the many hours of work and helpfuldiscussions contributedby Start Hefta, GottfriedFeistner, Kay Rutheffurd, Mitsuru Haniu, Jimmy Calaycay, Michael Ronk, Michael Davis, and Shane Rutherfurd. This work was supported in part by NIH grants CA37808, GM40673, DK33155, HD14900,and CA33572.
[20] S t r a t e g i e s f o r L o c a t i n g D i s u l f i d e B o n d s in P r o t e i n s
By DAVID L. SMITH and ZHONGRUI Z a o u Disulfide bonding in proteins is one of the most frequently encountered posttranslational modifications of proteins. Because of the important role played by disulfide bonds in establishing and maintaining the three-dimensional character of proteins, it is important to know whether a protein or peptide contains disulfide bonds, and, for proteins that contain more than two half-cystinyl residues, it is important to know which residues are joined by disulfide bonds. Although there is a good method for quantifying the number of disulfide bonds in protein, l the unambiguous determination of the locations of disulfide bonds continues to challenge protein chemists. Disulfide cross-linkages have often been located by cleaving a protein between the half-cystinyl residues (i.e., with disulfide bonds intact) and identifying the disulfide-containing peptides by their amino acid compositions or sequences. 2-4 An alternative method, which is based on the observation that disulfide bonds may be reduced sequentially under specific conditions, has also been used successfully. 5-7 Although most of our present understanding of disulfide bonding in proteins can be traced to the successful application of these methods, they are often inadequate for the most challenging problems. For example, disulfide-containing peptides can be identified by their amino acid composition or sequence only if they 1 T. W. Thannhauser, Y. Konishi, and H. A. Scheraga, this series, Vol. 143, p. 115. 2 p. E. Staswick, M. A. Hermodson, and N. C. Nielsen, J. Biol. Chem. 259, 13431 (1984). 3 T. W. Thannhauser, C. A. McWherter, and H. A. Scheraga, Anal. Biochem. 149, 322 (1985). 4 L. Haeffner-Gormley, L. Parente, and D. B. Wetlaufer, Int. J. Pept. Protein Res. 26, 83 (1985). 5 j. R. Reeve, Jr., and J. G. Pierce, Int. J. Pept. Protein Res. 18, 79 (1981). 6 W. R. Gray, F. A. Luque, R. Galyean, E. Atherton, R. C. Sheppard, B. L. Stone, A. Reyes, J. Afford, M. McIntosh, B. M. Olivera, L. J. Cruz, and J. Rivier, Biochemistry 23, 2796 (1984). 7 T. E. Creighton, this series, Vol. 107, p. 305.
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