Jul 15, 2016 - marked âadvertisementâ in accordance with 18 U.S.C. Section 1734 ...... Met - Ile - LyS - Pro - Phe - Phe - His - Ala - Leu - Ser - Clu - Lye. 1. 2. 1.
VOl. 263, No. 20, Issue of July 15,pp. 9589-9597,1988 Printed in U.S.A.
OF BIOLOGICAL CHEMISTRY THE JOURNAL 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc
Amino Acid Sequence of Thioredoxin Isolated from Rabbit Bone Marrow Determined by Tandem Mass Spectrometry* (Received for publication, February 24, 1988)
Richard S. Johnson$, W. Rodney Mathews$§,Klaus BiemannSIl, and Sarah Hopper11 ** From the $Department of Chemistry, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139 and the IlSchool of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
The amino acid sequence of the thioredoxin isolated observation that thioredoxin catalyzes the refolding of disulfrom rabbit bone marrow was determined chiefly by fide-containing proteins (12) is consistent with the high dehigh performance tandem mass spectrometry and fast gree of homology between the active sites of protein-disulfide atom bombardment mass spectrometry combined with isomerase and thioredoxin (13). manual Edmandegradation. The sequences of peptides The primary structures have been determined for thioregenerated by digestion with trypsin alone or in com- doxins from a wide variety of prokaryotic organisms including bination with Staphylococcus aureus protease V8 or the bacteria E. coli (14, 15) and C.nephridii (5, 16), the thermolysin were determined from their collision-in- bacteriophage T4 (17), the cyanobacterium Anabaena spheduced dissociation mass spectra. Alignment of these roides 7119 (18), and the photosynthetic bacteriaChromatiurn sequences and additional sequence information were uinosum (19), Chlorobiumthiosulfatophilum (20), Rhodoobtained from the collision-induced dissociation mass spirillum rubrum (21), and very recently Rhodobucter sphaespectra of peptides obtained from digestion of the intact roides Y (39), as well as for the m-type of thioredoxins from protein with S. aureus protease V8 and a-chymotrypsin. The resulting sequence of 104 residues is as fol- spinach chloroplasts (22). The complete amino acid sequence of thioredoxin of rabbit bone marrow reported here is the first lows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Glnto Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val-be determined for a mammalian thioredoxin. Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-LysMATERIALS AND METHODS’ Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-LysPhe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp-Fast Atom Bombardment Mass Spectrometry-FABMS2 of the Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lysproteolytic peptides was carried out on the first (MS-1) of two mass Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys-spectrometers of a tandem high resolution mass spectrometer (JEOL Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-LysHXllO/HX110) at an accelerating voltage of 10 kV and a resolution Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu. of 1:2200 and with 100-Hz filtering. For calibration, (CsI),Cs+ cluster
Thioredoxins are small proteins with a cystine disulfide/ dithiol active center which function in anumber of oxidationreduction reactions. While first shown to reduce methionine sulfoxide (1)and sulfate (2),thioredoxin was characterized as a hydrogen donor for ribonucleotide reduction in Escherichia coli (3). This role for thioredoxin was generally accepted until an alternate hydrogen donor protein, glutaredoxin, was discovered in a thioredoxin-defective mutant of E. coli (4).Further evidence that thioredoxin is not essential for ribonucleotide reduction is that neither the C-1 thioredoxin of Corynebacterium nephridii ( 5 ) nor the thioredoxin of rabbit bone marrow (6) is a hydrogen donor for the homologous ribonucleotide reductase. Thioredoxins are known to have other functions including the regulation of the activity of proteins (7, 8 ) and the binding of glucocorticoids by their receptors (9), as well as serving as a subunit of the bacteriophage T7 DNA polymerase (10) and as anessential component for the assembly of filamentous bacteriophages (11).The more recent * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 Present address: The Upjohn Co., Kalamazoo, MI 49001. 7 Recipient of Research Grants GM 05472 and RR00317 from the National Institutes of Health. ** Recipient of Research Grant PCM-7923540 from the National Science Foundation and the Office of Research, University of Pittsburgh.
ions were used. Single scans were acquired a t a rate to scan from m/z 200 to 3000 in about 2.5 min. The JEOLFAB gun was operated at 6 kV with xenon as the FAB gas. Tandem Mass Spectrometry-Tandem mass spectrometry was carried out by using all four sectors of the JEOL HXllO/HX110, an instrument of ElBlE2B2configuration. Collision-induced dissociations (CID) took place in the third field-free region, thus operating both MS-1 (EIB1) andMS-2 (E2B2) as double-focusing instruments. Helium was used as thecollision gas at a pressure sufficient to reduce the precursor ion signal to 50-30%. The CID mass spectra were recorded a t 3 0 - Hfiltering, ~ and the scan rate was the same as used for FABMS. Generally, for CID spectra, the resolution of MS-1 was adjusted to transmit only the ‘*C-speciesof the (M + H)+ ion to be analyzed. MS-2 was usually operated a t a resolution of1:lOOO; however, for the larger peptides ((M + H)+ > ZOOO), the resolution was 1500. MS-2 was calibrated with a mixture of CsI, NaI, KI, andLiCl (23). The FAB and CID mass spectra shown in Figs. 2-4 are raw profile data of single scans and were recorded with a JEOL DA5000 data system. RESULTSANDDISCUSSION
Prior to the installation of the JEOL HX110/HX110 tandem mass spectrometer at Massachusetts Institute of Tech-
’
Portions of this paper (including part of “Materials and Methods,” Tables I, 111, V, VII, and IX-XI, and Scheme I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. The abbreviations used are: FABMS, fast atom bombardment mass spectrometry; CID, collision-induced dissociation; HPLC, high performance liquid chromatography; Xle, leucine or isoleucine; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone.
9589
9590
Amino Acid Sequence
of Thioredoxin from
nology, 24 cycles of gas-phase Edman degradation were carried out on the intactprotein. However, position 14 was later shown from CID mass spectral data to have been incorrectly assigned as phenylalanine. Additional sequence information was obtained by subjecting HPLC fractions of proteolytic peptides to manual Edman degradations, followed byFABMS to determine the masses of the truncated peptides. The difference in molecular weight of each peptide in the mixture before and after each step of Edman degradation indicates the successive N-terminal amino acids in each peptide. With this technique, the isomeric amino acids leucine and isoleucine cannot be distinguished. Although the amino acids glutamineand lysine are of the same nominal mass, this is usually not aproblem when found in tryptic peptides because lysine is a tryptic cleavage site. Furthermore, the presence of lysine manifests itself by the addition of 135 daltons due to the reaction of the eaminogroup with phenyl isothiocyanate during the first Edman step (this differentiation cannot be made for the N-terminal amino acid because it is lost in the first step). The information thus generated will hereafter be referred to as FABMS/Edman data, and theresults of these experiments for three proteolytic digests are listed in Table I. In contrast to conventional Edman sequencing, tandem mass spectrometric sequencing of proteins does not require complete separation of each peptide since the protonated molecules, (M H)+, for each peptide, formed by FAB ionization, can be selected by the first double-focusing mass spectrometer (MS-1) and caused to collide with an inert gas (helium) inthe region between MS-1 and the second doublefocusing mass spectrometer (MS-2). The masses of the product ions formed by fragmentation upon collision are then determined by properly scanning MS-2 ("CID mass spectrum"). All of these ions must, of course, result from fragmentation of only the (M H)+ ion selected by MS-1 (24, 25). The strategy for sequencing proteins by tandem mass spectrometry (19, 26) typically involvescleavage with a sitespecific protease, e.g. trypsin, and partial fractionationof the resulting peptides by reverse-phase HPLC. The molecular weights of the peptides present in each fraction are then determined by FABMS, and the sequences are subsequently deduced from the fragmentations observed in the CID mass spectra. In some cases, tryptic HPLC fractions may be cleaved further with a different enzyme, thus generating smaller peptides for collisional activation. In order to obtain overlapping sequences that allow for the proper arrangement of the sequenced tryptic peptides, the intact protein is degraded with a different enzyme, e.g. S. aureus protease V8, or a-chymotrypsin, and the resulting peptides are then sequenced. Frequently, the molecular weights of these peptides alone permit 6 the unique alignment of the tryptic peptides. 7 This general approach was used to determine the primary structure of the thioredoxin isolated from rabbit bone marrow. The protein was digested with trypsin, and the peptides were separated into eight fractions by reverse-phase HPLC (Fig. 5 7 1). The molecular weights of the peptides in each fraction were determined by FABMS (Table 11),and CID mass spectra were measured for nine of the (M + H)+ ions observed. The sequences summarized in Table I1 were deduced from these data. Table I11 lists the fragment ion masses and assignments for these CID spectra. The sequence deduced from the CID mass spectrum of (M H)+ = m/z 1366.8 corresponds to cycles 8-20 of the gas-phase Edman data, except that leucine rather than phenylalanine was found at position 7 of this tryptic peptide. This interpretation is supported on the basis of fragment ions differing by 113 daltons, the residue mass of leucine and isoleucine; and the mass of the w n ion for this
Rabbit Bone Marrow
+
1
+
+
20
10
E l u t i o n T i m e (rnin) FIG. 1. HPLC chromatogram of tryptic digest of S-carboxymethylated rabbit bone marrow thioredoxin. The markings indicate the fractions collected for analysis by FAB and CID mass spectrometry.
TABLEI1 (M + H)+ ions of tryptic peptides (M + H)+
HPLC fraction
Position"
Sequenceb
2 3-7 604.1 QIESK 635.4 4 36-40 MIKPF 2 1-7 831.5 VKQIESK VGEFSGANK 908.6 85-93 3 1015.6 96-104 LEATINELL 1165.8 3 85-95 1206.7 72-80 C*MPTFQFFK 1272.8 6 94-104 EKLEATINELL 72-81 1334.8 6 8-20 SAFQEVLDSAGDK 1366.8 5 MIKPFFHALSEK 36-47 1447.9 21-35 1740.5 85-104 2162.3 8 8-35 3088.4 8 Positions are those corresponding to thefinal structure. * Sequences were determined from CID mass spectral data; CID spectra were not measured if no sequence is shown. In some instances where amino acids could not be assigned from individual CID spectra, they were deduced from the CID data of overlapping peptides. C* indicates carboxymethylated cysteine.
position allowed for the differentiation of these two amino acids. The incorrect assignment was probably due to the fact that the retention times for the phenylthiohydantoin-deriva-
Amino Acid Sequence
of Thioredoxin from
tives of leucine and phenylalanine were similar under the HPLC conditions used. FABMS of HPLC fraction 8 revealed only two (M H)+ ions, both of relatively high mass and neither of which was sufficiently abundant to obtain their CID spectra. This fraction was therefore subjected to a S. aureus protease V8 digest, followed by HPLC fractionation in order to obtain smaller peptides more amenable to collisional activation. As a result, eight peptides were observed and are listed in Table IV. The CID spectra of the tryptic/S. aureus protease peptides of (M + H)+ = m/z 547.1 and 1122.4 led to the conclusion that the peptide of (M H)+ = m/z 2162.3 from fraction 8 (Fig. 1) was composed of the two tryptic peptides of (M + H)' = m/z 908.6 and 1272.8 (see Table 11) resulting from incomplete cleavage of the Lysg3-G1ug4bond. More specifically, upon collisional activation, the tryptic/S. aureus protease peptide of (M H)+= m/z 547.1 revealed the sequence Ala-Thr-Xle-Asn-Glu, and that of m/z 1122.4 had the seThe diquence Phe-Ser-Gly-Ala-Asn-Lys-Glu-Lys-Xle-Glu. and tripeptides Xle-Xle and Val-Gly-Glu, which would make up the remainder of the original peptide of fraction 8 ( m / z 2162.3), were not observed presumably because of the high matrix background at lower mass in FABMS. Although these results do not provide additional sequence information in these regions, the partial cleavage peptide indicates that the tryptic peptide of (M H)' = m/z 1272.8 is preceded by that of m/z 908.6. The tryptic/S. aureus protease peptides of (M + H)+ = m/z 581.1, 1215.4, and 1329.6 (Table IV) were also sequenced from their CID spectra, and the data are listed in Table V. From this, it was determined that the original peptide of fraction 8, (M H)' = m/z 3088.4, was derived from incomplete tryptic cleavage of the LysZ0-Leuz1bond and was later shown to encompass positions 8-35 of the protein sequence. Again, these results provided overlap information, but additional regions of the protein were also sequenced. The CID H)' = m/z 1215.4 provided the mass spectrum of (M sequence of the active-site region (-Trp-Cys-Gly-Pro-CysLys), butthe portion N-terminal to the trmtophan could not be reliably determined from this spectrum. Interestingly, the remaining three tryptic/S. aureus protease peptides of (M + H)+ = m/z 981.5, 1350.5, and 1640.4 are not derived from any peptide observed in the FAB mass spectrum of tryptic HPLC fraction 8. The most likely explanation is that fraction 8 contains three peptides, those of (M + H)' = m/z 2162.3 and 3088.4, plus an additional one, encompassing positions 48-71, of high hydrophilicity which did not readily ionize by FAB in the presence of the more
+
+
+
+
+
+
Rabbit Bone Marrow
9591
hydrophobic peptides (27). The CID mass spectral dataof the tryptic/S. aureus protease peptide of (M H)' = m/z 981.5 is shown in Table V. That of (M H)' = m/z 1350.5 was not sufficiently abundant for collisional activation, and the CID mass spectrum of the tryptic/S. aureus protease peptide of (M + H)' = m/z 1640.4 revealed only partial sequence information. These two peptides were, however,produced in abundance when the protein was digested with S. aureus protease alone and gave good CID spectra (Table VI). In order to properly arrange the observed tryptic and tryptic/S. aureus protease peptides as well as to obtain sequence information for regions not covered by these digests, the Scarboxymethylated protein was cleaved with S. aureus protease V8 and partially fractionatedby HPLC, and the molecular weights were determined by FABMS (Table VI). The partial FAB mass spectrum of one of the HPLC fractions is
+
+
TABLEVI (M + H)+ions of S. aureus V8 protease peptides (M + H)' Position" Sequenceb ATINE 98-102 547.0 1-5 616.1 VKQIE 88-94 FSGANKE 752.1 6-12 796.1 SKSAFQE 70-771011.3 VKC*MPTFQ 47-551109.3 KFNNVVFIE 88-97 1122.3 FSGANKEKLE 78-871167.3 FFKKGQKVGE 13-251329.4 VLDSAGDKLVVVD 56-671350.3 VDVDDC*KDIAAE 56-691640.3 VDVDDC*KDIAAEC*E 70-872159.8 VKC*MPTFQFFKKGQKVGE 26-46 2515.7 3826.5 a
13-46
Positions are those corresponding to the final structure.
'Sequences were determined from CID mass spectral data; CID spectra were not measured if no sequence is shown. In some instances where amino acids could not be assigned from individual CID spectra, they were deduced from the CID data of overlapping peptides. C* indicates carboxymethylated cysteine.
"
~~
(M+H)+
= m/z
1109.3
/
2 n
TABLEIV (M + H)' ions of tryptic/S. aureus protease peptides (M + H)' Position" Sequenceb 98-102 48-55 26-35 13-25
547.1 581.1 981.5 1122.4 1215.4 1329.6 1350.5 1640.4
8-12 88-97
z m
(M+H)+
=
ATINE SAFQE FNNVVFIE FSGANKEKLE FSATWC*GPC*K VLDSAGDKLVVVD
m/z
2159.8
\I
56-67 56-69 ~~
~
Positions are those corresponding to the final structure.
* The sequences shown were deduced from their CID mass spectra.
No CID spectrum was obtained for (M + H)' = m/z 1350.5. and that of (M + H) = m/z 1640.4 was too poor in quality (low signal-tonoise ratio) to determine its sequence. In some instances where amino acids could not be assigned from individual CID spectra, they were deduced from the CID data of overlapping peptides. C* indicates carboxymethylated cysteine. +
1060
1100
1140
1180
2100
2140
m/
2180
2
FIG. 2. Partial FAB mass spectrum of HPLC fraction 6 of S.aureus protease V8 digestion of S-carboxymethylated rabbit bone marrow thioredoxin. Two peptides of (M + H)' = m/z 1109.3 and 2159.8 were observed in this fraction.
Amino Acid Sequence Thioredoxin of
9592
I
-,jd
c!,!~,
4 ) ~
i : , 6,)' 'I
:uu
b,.Lm
i
I#)_
'
1 1
I
FIG. 3. CID mass spectrum (utilizing all four sectors of a high performance tandem mass spectrometer) of (M H)+ = m/z 1109.3 (see Fig.2). Ions labeled NN and NNVdenote internal fragment ions; V, K, and F are immonium ions of valine, lysine, and phenylalanine; and -E and - V are ions derived from elimination of the side chains of glutamic acid and valine. The remaining ion types are depicted in Scheme I.
+
shown in Fig. 2, which reveals the presence of two peptides of (M H)' = m/z 1109.3 and 2159.8. The CID mass spectra of these two peptides are shown in Figs. 3 and 4. The sequence deduced from the former represents the sequence determined from the CID mass spectrum of the tryptic/S. aureus protease peptide of (M H)' = m/z 981.5 (Table IV), with an additional lysine at theN terminus. Thus, thispeptide ( m / z981.5) is C-terminal to a tryptic cleavage site. Fig. 3 also contains a product ion at m/z 906.4 corresponding to da, demonstrating that position 8 is isoleucine (38)3(for the fragment ion notation, see Scheme I). Fig. 4 shows one of the larger peptides for which useful CID mass spectral data have been acquired for a peptide of unknown sequence. Although smaller peptides within the region covered by this peptide more clearly delineate the sequence, this spectrum provides further proof of the sequence and gives unambiguous overlap information. The CID mass spectral data for these and other peptides that either provide overlap or additional sequence information are listed in Table VII. CID mass spectra were not obtained for (M + H)+ = m/z 2515.7 and 3826.5.However, the masses alone were sufficient to provide overlap information demonstrating that Lys35 is followedby Met36, and in the final structure, thesepeptides encompass positions 26-46 and 1346, respectively. At this point in our work, nearly the entire structure shown in Fig. 5 had been determined. Since the t-amino group of lysine reacts with phenyl isothiocyanate during the first manual Edman degradation cycle, all of the glutamines and lysines had been differentiated from the earlier FABMS/Edman data
+
+
Martin, S. A,, and Biemann,K., 34th AnnualConference on Mass Spectrometry and Allied Topics, Cincinnati, OH, June &13,1986, p. 854.
from Rabbit Bone Marrow (Table I). Also, all of the leucines and isoleucines had been differentiated from the masses of d, (38)3 and w n (28) ions, except for the C-terminal position of the protein. However, there were a few points that needed clarification. First, positions 26-29 and 68-69 needed sequencing; second, further overlap covering positions 47-48, 55-56, and 69-70 was required. For this purpose, a third portion of S-carboxymethylated rabbit bone marrow thioredoxin was digested with m-chymotrypsin. Again, the proteolytic fragments were partially fractionated and analyzed by FABMS. The CID spectra of 13 chymotryptic peptides were obtained,and the resultsare depicted in Fig. 5. Most of these peptides covered regions already sequenced; those that were relevant to theopen questions discussed above are listed in Table VIII, and their CID mass spectral data are shown in Table IX. The peptide of (M H)' = m/z 578.2 was shown to have the sequence Val-ValVal-Asp-Phe, thus demonstrating that phenylalanine is at position 26. Likewise, the peptide of (M H)' = m/z 464.2 was shown to have a C-terminal sequence of Thr-Trp; however, the N-terminal portion, of residue mass (-NH-CHR2'CO-NH-CHRZ8-CO-)158 daltons, could not be confidently sequenced from this CID mass spectrum. Hence, positions 27-28 could contain only either Gly + Thr (57 101 daltons) or Ala Ser(71 + 87 daltons), but still remained unseH)' = m/z 978.4 provided quenced. The peptide of (M overlap for positions 47-48. Finally, the C-terminal peptide representing positions 89-104of (M + H)' = m/z 1729.9 (which encompasses a previously sequenced region and therefore is not shown in Table IX) was found to be present as a single component in the latest eluting HPLC fraction. This provided an opportunity to differentiate the C-terminal Xle by amino acid analysis of this fraction (Table X). Not only were the results consistent with the sequence deduced from the CID mass spectrum, but they also clearly indicated the presence of 3 leucines and 1 isoleucine. Since the mass spectral data demonstrated that this peptide contains 2 leucines, 1 isoleucine, and 1 undifferentiated Xle at the C terminus, the latter must be leucine. The final experiment that completed the sequence involved a second tryptic digest of the intactprotein, followed byHPLC fractionation. From the earlier tryptic digest, it was found that thepeptide encompassing positions 48-71, which did not readily ionize by FAB, eluted late in the chromatogram. Therefore, fractions corresponding to 7 and 8 of Fig. 1 were cleaved further with thermolysin, separated by HPLC, and analyzed by FABMS and tandem mass spectrometry. Much of the resulting data covered positions already sequenced and are not reproduced here. Three CID mass spectra (Tables VI11 and XI) contained the relevant sequence data.The
+
+
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+
+
~~
S'ol-Lys-Cys'-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys-Gly-Gln-Lys-Val-Gly-Glu
FIG. 4. CID mass spectrum (utilizing all four sectors of a high performance tandem mass spectrome- : ter) of (M + H)+ = m/a 2159.8 (see Fig. 2). Ions PT,PTFQ, PTFQF, and QF are internal fragmentions, and those labeled K and F are immonium ions of lysine and phenylalanine. The remaining ion types are depicted in Scheme I. Assignments were made in part based on the CID spectra of smaller peptidesthat encompass the same region of the protein (see text). Cy,", carboxymethylated cysteine.
100 200 300 400
500 500 i 0 0 800 900 1000 1 1 00 1200 1300 1400 4
2
1500 1600 1700 1' 800 19X 200C 21 30
4-63
Amino Acid Sequence of Thioredoxin from
Rabbit Bone Marrow
9593
Val Lys Gln Ile Glu Ser Lys Ser Ala Phe Gln GluLeu ValAsp Ser Ala Gly Asp Lys a ” ”
20
7
0
7
a
7
-
”-
a
a
0
Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys 4Pro Phe 0 -
7
”
FIG. 5. Amino acidsequence of thioredoxin from rabbit bone marrow. a, tryptic peptides; b, peptides resulting from digestion of tryptic HPLC fraction 8 with S. aureus protease V8; c, peptides generated by digestion of the intact protein with S. aureus protease; d , a-chymotryptic peptides; e, peptides resulting from digestion of late eluting tryptic HPLC fractions with thermolysin. Heavy underlining, sequence derived from CID mass spectra; half-arrows, amino acids removed by manual Edman step. Overlining of the first 24 amino acids indicates sequence determined by automated gas-phase Edman degradation. The gap in the ouerlining at position 14 indicates an error in the gas-phase Edman data that was corrected by tandem mass spectrometry (see text).
a
-
a
0
a
L
Phe His Ala Leu Ser
Glu
Lys
Phe
Asn
Asn Val Val Phe Ile Glu Val Asp Val Asp Asp 6 0
”
7
0
a”” a 0
80 cys Lys Asp Ile Ala Ala Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys ””
0 -
*
a-
-
.-
-c
--
Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys GluLeuLys Glu Ala Thr Ile
*-
*
7
0 0 - ”
7
Asn
” -
----
100
””
a
*-
-
Glu Leu Leu
TABLEVI11 (M + H)’ ions of chymotryptic peptides and trypticlthermolytic peptides
104
a thioredoxin of mammalian origin. However, as indicated previously by their amino acid compositions, animal thioredoxins are unique in that they contain no arginine and have (M + H)+ Position” Sequenceb cysteines in addition to thetwo thiols at theactive site of all thioredoxins (6,29-31). Two thioredoxins, C-1 and C-2, have Chymotryptic peptides 464.2 SATW which contains been isolated from C. nephridii, and the latter, VVVDF 578.2 3 cysteine residues (16), is the only prokaryotic thioredoxin 978.4 FHALSEKF known to have more than two thiols. The sequence data 89-104 1729.9 C-terminal peptide presented in Fig. 5 show that the bone marrow thioredoxin Tryptic/thermolytic peptides VVVDFS contains neither arginine nor tyrosine and has 5half-cystines 665.3 IAAEC*EVK 920.5 which include the active-site disulfide in the tetrapeptide 1208.5 IEVDVDDC*KD Cy~~l-Gly-Pro-Cys~~and the cysteines at positions 61, 68, a Positions are thosecorresponding to thefinal structure. and 72. The same active-site tetrapeptide sequence has been *Sequences were determined from CID mass spectraldata. In identified in all thioredoxins examined with the exception of some instances where amino acids could not be assigned from individual CID spectra, they were deduced from the CID data of overlap- phage T4 thioredoxin (17) and the C-2 thioredoxin of C. nephridii (16), which have the sequences -Cys-Val-Tyr-Cysping peptides. C* indicates carboxymethylated cysteine. and -Cys-Ala-Pro-Cys-,respectively. The thiols of glutaredoxpeptide of (M + H)+ = m/z 665.3 was shown to be Val-Val- ins are arranged similarly, i.e. the glutaredoxin of E. coli (32) Val-Asp-Phe-Ser, thus demonstrating that sequence 27-28 is has only the one active-site dithiol, whereas the glutaredoxin Ser-Ala. The CID mass spectra of the peptides of (M H)+ of calf thymus (33) and the glutaredoxin-like pig liver thiol= m/z 1208.5 and 920.5 provided overlapping sequences for transferase (34) contain an additional dithiol. In both of the positions 55-56 and 69-70 and demonstrated that positions latter proteins, the two active-site cysteines are separated by 68-69 have the sequence Cys-Glu. two amino acids and the other half-cystine pair by three. Whereas this dithiol configuration resembles that of the bone CONCLUSION marrow thioredoxin, information on the primary structure of The complete amino acid sequence of the thioredoxin of other animal thioredoxins now is limited to the partial serabbit bone marrow presented here is the first determined for quence determined for the rat hepatoma thioredoxin (35). In ~~
~
~~
+
Amino Acid Sequence of Thioredoxinfrom Rabbit Bone Marrow
9594 E. coli C. nephridii Spin. chlor. Rabbi t
E. coli Set he Asp Thr sp Va C. nephridii
Spin. chlor. Rabbit
Asn UTrp G lLYSnGly Glu Ser WPhe G
Leu L s
Leu i Val Glnn
Phe GlnGlu Val L e u Asp Ser Ala 10
E. coli C. nephridii Spin. chlor.
REFERENCES 1. Black, S., Harte, E.M., Hudson, B., and Wartofsky, L. (1960)J. Biol. Chem. 235,2910-2916 2. Wilson, L. G., Asahi, T., and Bandurski, R. S. (1961)J. Biol. Chem. 236,
_"_"
1 R39-1 Q39 " "
3. Laurent, T. C., Moore, E. C., and Reichard, P. (1964)J. Biol. Chem. 239, 3436-3444 4. Holmgren, A. (1976)Proc. Natl. Acad. Sci. U. S. A. 73,2275-2279 5. Meng, M., and Hogenkamp, H. P. C. (1981)J.Biol. Chem. 266,9174-9182 6. Hopper, S., and Iurlano, D. (1983)J. BWL Chem. 258,13453-13457 7. Holmgren, A. (1979)J. Biol. Chem. 264,9113-9119 8. Buchanan, B. B. (1980)in The Enzymology of Post-translational Modifications off'roteins (Freedman, R. B., and Hawkins, H. C., eds) Vol. 1, pp. 345-362,Academic Press, New York 9. Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983)J.Bzol. Chem. 268,13658-13664 10. Mark, D. F., and Richardson, C.C. (1976)Proc. Natl. A c d . Sci. U. S.A. 73,780-784 11. Russel, M., and Model, P. (1985)Proc. Natl. Acad. Sci. U. S. A. 82,29-33 12. Pigiet, V. P., and Schuster, B. J. (1986)Proc. Natl. Acad. Sci. U. S. A. 83, 7643-7647 13. Edman. J. C.. Ellis. L.. Blacher. R.W.. Roth. R.A,. and Rutter. W. J. (19&) Nature 317, 267-270 ' 14. Holmgren, A. (1968) Eur. J. B~ochem.6,475-484 15. Lim, C.-J., Geraghty, D., and Fuchs, J. A. (1985)J. Bacteriol. 163, 311-
Rabbit
E. coli C. nephridii Spin. chlor. Rabbit 40
E. coli C . nephridii Spin. chlor. Rabbit E. coll C. nephridii Spin. chlor. Rabbit
E. c o l i
316
C. nephridii Spin. chlor. Rabbi t
16. Lim.C.-J.. Fuchs. J. A,. McFarlan. S. C.. and Hoeenkamu. H. P. C. (1987) , , J.'Biol. Chem. 262,12114-12119 ' 17. Sjobetg, B.-M., and Holmgren, A. (1972)J. Biol. Chem. 247,8063-8068 18. Gleason. F. K.. Whittaker. M.M.. Holmmen. A., and Jornvall, H. (1985) . . J. Bwl. Chem. 260,9567-9573 19. Johnson, R. S., and Biemann, K. (1987)Biochemistry 26, 1209-1214 20. Mathews, W. R., Johnson, R. S., Cornwell, K. L., Johnson, T. C., Buchanan, B. B., and Biemann, K. (1987)J.Biol. Chem. 262,7537-7545 21. Johnson, T. C., Yee, B. C., Carlson, D. E., Buchanan, B. B., Johnson, R. S., Mathews, W. R., and Biemann, K. (1988)J. Bacteriol. 170, 24062408 22. Maeda, K., Tsugita, A,, Dalzoppo, D., Vilbois,F., and Schurmann, P.(1986) Eur. J. Biochem. 164,197-203 23. Sato, K., Asada T., Ishihara, M., Kunihiro, F., Kammei, Y., Kubota, E., Costello. C. E.. Martin. S. A,. Scohle. H. A.. and Biemann.. K. (1987) . . ~ n a l chm. . 59,1652-i659 ' 24. McLaffertv. F. W. (ed) (1983) Tandem Mass Swctrometrv. John Wilev & . . . . ". Sons, New York 25. Martin, S. A,, and Biemann, K. (1987)Int. J. Mass Spectrom. Ion Proc. 78,213-228 26. Biemann, K., Martin, S. A., Scoble, H. A., Johnson, R. S., Papayannopoulos, I. A,, Biller, J. E., and Costello, C. E. (1986)in Mass Spectrometry in the Analysis of Large Molecules: Proceedings of the Texns Symposium on Mass Soectrometrv (McNeal.. C... ed) UP. 131-149. John Wiles & Sons Ltd., Sissex, Englind 27. Navlor. S.. Findeis. A. F.. Gibson. B. W.. and Williams. D. H. (1986) . . J. Am. %&m. SOC. 108,6359-6363 28. Johnson. R. S.. Martin. S. A,. Biemann. K.. Stults.. J. T.., and Watson. J. T. (1981)Anhi. Chi;. i9;i621-2625 ' ' 29. Luthman, M., and Holmgren, A. (1982)Biochemist 21,6628-6633 30. Enestrom. N.-E.. Holmmen. A,. Larsson.. A... and foderhall. S. (1974)J. Bioi. Chrn. 249,205-210. 31. Herrman, E. C., and Moore, E. C. (1973)J.Biol. Chem. 248,1219-1223 32. Hoog, J.-O., Jornvall, H., Holmgren, A,, Carlquist, M., and Persson, M. (1983)Eur. J. Biochem. 136,223-232 Hoog, J.-O., Jornvall, H., Holmgren, A., and Luthman, M. 33. Klintrot, I."., (1984)Eur. J. Biochem. 144,417-423 34. Gan, 2.-R., and Wells, W. W. (1987)J. Biol. Chen. 262,6699-6703 35. Guevara, J., Jr., Moore, E. C., and Ward, D.N. (1983)in Thioredoxiw: Structure and Function (Gadal, P., ed) pp. 79-83,Centre National de la Recherche Scientifique, Paris 36. Alberts, B., Bray, D.,.Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1983)Molecular Bzology of the Cell, pp. 19-20,Garland Puhlishlng Inc., New York 37. Tarr, G. A. (1977)Methods Enzymol47,335-357 38. Johnson. R. S. (1988) . . Ph.D. dissertation. Massachusetts Instituteof Technolo& 39. Clement-Metral,J. D., Holmgren, A,, Cambillau, C., Jornvall, H., Ecklund, J. Biochem. 172,413-419 H., Thomas,D., and Lederer, F. (1988) Eur. _
B. coli Glu Phe Leu Asp Ala AsnLeu Ala C. nephridii Asp Trp Ile Lys
Rabbit
i.e. essentially half of the residues of all three proteins are identical. That chloroplast thioredoxin would resemble bacterial thioredoxin far more than a mammalian thioredoxin may not be unexpected given the commonly held view that chloroplasts are of prokaryotic origin (36).
Ser Ala
Ile Asn Glu k u k u 100
104
FIG. 6. Comparison of amino acid sequences of E. coli thioredoxin (14,15),C - l thioredoxin of C.nephridii ( 5 ) ,mb-type thioredoxin from spinach chloroplasts (22), and rabbit bone marrow thioredoxin. Identical residues are boxed.
the N-terminal region of the molecule, 28 of 35 amino acids are identical in the rat and rabbit proteins. These include valine at the N terminus and the heptapeptide Trp3'-CysGly-Pr~-Cys-Lys-Met~~- atactive the site. Whereas data obtained by amino acid analysis indicate the presence of 6 cysteines in the hepatoma thioredoxin (31), only 2 of these are located in the segment which was sequenced. Comparison of the primary structures of the thioredoxins from rabbit bone marrow, E. coli, C. nephridii (C-1), and spinach chloroplasts (type mb) with these proteinsaligned by the active site (Fig. 6) shows the most highly conserved region of the molecule to extend from to Pro39 (rabbit bone marrow numbering). The sequence of this 16-residue segment in the E. coli and C. nephridii (C-1) proteins differs by only two amino acids from chloroplast thioredoxin and three from rabbit thioredoxin. However, 18 of the first 23 residues from the N terminus and 55 of the last 65 C-terminal residues of the rabbit protein have no sequence identity with any of the other threethioredoxins. In contrast, the complete amino acid sequences of the E. coli, C. nephridii (C-1), and spinach chloroplast (type mb) thioredoxins are remarkably similar,
~
~~~~~
~~~
I
Amino Acid Sequence of Thioredoxin from Rabbit Bone Marrow
9595
Supplementary material t o m i n o Acid Sequence of Thioredoxin Isolated Prom Rabbit BOne Marrow D e t e m i n e d by Tandem Mas- Spectrometry
UATERIAlS AND METHODS
-
P u r i f i c a t i U thioredoxin ThioredoXin was purified from the bone m~rrow old noma1 rabbits an previously described (6).
Of eight weeks
-
"hioradoxin Reduction and S-carboxvmathvlation of t h i o r a was reduced and S-carboxymethylated as previously described (6).
rH+H)*
-
-on of DeDtides for mass eoectroS-carboxymathylated rabbit bone marrow thioredoxin (360 u g ) vas digested with trypsin (50:l substrate:enzyne) for 2 hrs in 0.1 R NHHm and 0.1 mM CaC1, at pH 8.5 and 37 * C. The tryptic digest was partially hac:ionated by reversed phase high performance liquid Ehr'OmtOgraphy (HPLC). Fraction eight was further c l e w e d (strain with V8) protease (50:l substrata:anayme) for four Intact S-carboxymethylatsd hours in 0.1 I4 NH4HC0 a t 37 * C and pH 8.0. thioredoxin 1120 Y 91 'was cleaved with 5 . oureue protease under thesame conditions and fractionated by reversed phase HPLC. Another 120 Y g of the S-carboxymethylsted protein was treated with ~-chy.lotrypsin using the same conditions described for the tryptic digest. Finally, another 80 v g of S-carboxymethylated thioredoxin was again cleaved with trypsin and partially separated by HPLC. The later eluting fractions were digested further with the endopeptidaee from R W i l l u s tharmoorotolvticus (50:l substrate:enzyme] for 1 hr in the tryptic buffer at 45 .c.
604.1
=
431.2 502.3 476.3 460.2 4 Ila 2 213.9 242.0
Y Ztl
5 Gln 1
-
363.0 146.9 234.0 347.1 3 2 G1U - Ser 3 4 343.1 371.0 458.2 285.3
159.1 530.3 504.3 488.2 4
Y Ztl
5 Wet 1
-
m' =
-
Ile
2 216.9 244.9
b
391.1 375.1 3 Lyys 3
263.1 2 Pro 4 442.1 470.1
-
373.1
658.3 530.1 758.3 630.3 502.1 732.5 604.3 715.3 587.3 716.3 588.3 6 5 Lys Gln 2 3
Ztl 7 Val 1
-
-
b d
227.8
-
604
Y
635 831 908 1015 1206 1272 1366 1447
PI1
5 . ="reo"*
1 Ly6 7
389.3
332.2
373.3 4 Gly 6 549.2 577.0
316.1 3 Ala 7 620.3 648.2
4 A m 6 614.2 642.4
677.2 778.2 752.4
-
Y 9
Leu
1 1
1 b
0
1 0 1 2
156.8
285.9
520.2
773.4 7
702.0 244.8 601.3 6 5 Thr Ile 4 5
6
Phe
-
ser 5
4
=
+ -
902.0 8 Glu 2 500.1 214.9 242.8
-
-
146.8
2 A611 8 734.2 762.2
-
1 LY L
3 2 Glu Leu 7 8 743.0 856.4 771.2 884.4
1 Leu 9
-
-
Ala
3
-
-
313.9
-
415.3
528.1
871.3
784.0 843.3
914.3
Ztl 9
-
'.yC 1
0 0
b
1
-
-
,
771
0 0
-
1206.7 968.3 1071.3
2
-
260.8 243.8
=
+ -
1 1
736.2 7 Glu 3
200.8
443.1 502.0 476.1 459.0 460.0 5
649.2 623.4 606.5 607.5
800.2 801.2 6 Thr 4
(strain V81 DrOtea*e
All Val-LyS SeL-LYS-SeT Gln/Lys Phe-Ser-Gly-Ala-Aan VI1
-
793.2 8 Gly 2
Y 547 616 796 1109 1122 1640
2 Ser 6 657.3 685.3
-
E
Diqest
Gln/Lyr net Val-Lys-Gln Val-Gly-G1U Xle-G1U-Ala-Thr Cya*-net-PrO-Thr Glu-Lys-Xle Ser-Ala net-xle
233.8 146.8
3 Glu 5 570.6
Ile 4 441.1 598.3 469.1 413.0
356.3
835.4 809.3
b
in unsaauanced m r t i o n
TrVDtiC
363.3
531.2
FAms/Edman data.
seauenceq
287.9
476.1
m+ = w
9 Val 1
0'
431.0
460.1 4
-
T h e m o l y s i n and --chymotrypsin was obtained from Sigma Chemical Materiala CO., trypsin ITPCK treated) was purchased from Cooper Biomedical, and StaDhYloCOCCal aureus (strain V81 protease vas from Miles Scientific, mc.. Trifluoroacetic acid, cyanogen bromide, phenyliSDthioCy.n.te, heptane, pyridine, dithiothreitol. dithioerythritol, and ethyl acetatewere obtained from Pierce Chemical Company.
Number of lysines remaining
1 Phe 5
-
&lL.5
Y
NHt-Terminsl
1 Lya 5
=
IH+nl+
Amino acid anslvsis - Samples were hydrolyzed at 110 'C for 24 hr in an evacuated 10 x 7 0 m ignition tube containing 0.1 m 1 of constant-boiling HCI. Amino acid analysis was performed on a Dionex 0-500 amino acid analyzer.
288.3
-
b d
The fractionated proteolytic peptide mixtures were dissolved in glycerol at a concentration of 0.5-1.5 mol/r I. About 0.5 Y 1 of this solution vas placed on the sample probe tip and 0.5 Y 1 of 30% aqueous acetic acid alone, or in combination with a eutectic mixture of d i t h i o t h c e i t o l / d i t h i o a r y t h r i t o l (5:1), was mixed With the sample on the probe tip. \
Table I:
The m/r values of ions for Cys* corresponds to
Table 111: C I D ma68 spectral data for tryptic peptides. particular ion series (see Scheme I) are listed by rows. carboxymsthylated cysteine.
(800 u g )
1029.3 8 Met 2 264.6 292.6
7 Pro 3
-
-
490.8
495.0 716.4 569.1 441.3 293.8 146.7 552.0 700.3 553.0 277.7 425.2 5 4 3 2 1 Phe Gln - Phe Phe Lys 5 6 7 8 9 885.2 1032.1 638.0 766.0 1060.2
-
-
-
-
w* a - C h m o t m D t i C Disest
1169.3 978 1020
Phe-His-Ala-Xle Gln/Lys-Lys-Gly
1 1
1127.5
Z+l
11 G1u 1 ' X l e indicates
leucine or isoleucine; Cys'
-
10
LYS 2
-
indicates carbxymathylated cysteine. b d
257.6
9 Leu
3b2.8 371.0
1116.6 987.3 1305.5 1234.4 1087.2
13
ser 1 b
+ -
1263.5 1192.3 1045.3 1 2 1 1 1 0 Ala Phe - Gln 2 3 4
-
158.5 =
8
-
3
Y
Btl
773.1
1015.2
Y
-
305.6
GhI
-
4 472.3 499.9 414.3
858.1 959.1 933.1 9 G1U
-
5 562.7
7
Ala
6
-
Thr 6 644.2 672.0
5 542.9 571.0
760.1 773.2 830.1 804.2 788.1 8 Val 6
-
645.9 731.4 705.0 688.2 7
Leu 7
662.0 890.0 775.2
5 Ile 7 757.2 785.2 729.5
-
4 3 2 As" G1U - Le" 8 9 10 871.5 1000.2 1113.1 899.1 1028. 4 1141.5 828.3 942.5 1071.3
-
444.3 618.1 591.9 477.2
6 Asp 8
-
390.0
461.2 4 5 Ser Ala 9 1 0 1
-
977.3
1 Le" 11
-
318.7
261.6
2 ABp 2 1 1192.3 1105.4 1220.3 3
-
Gly 1 1
lri17.9 601.7
Y Z+l
1 2 Met 1 244.5
b d
1271.5 1032.3 1342.5 1229.5 1004.1 1316.5 1203.3 1075.3 978.2 1187.3 962.4 9 1 1 1 0 8 Ile LyS - Pro Phe 2 3 4 5 216.6 588.9 372.7 470.3
-
-
417.0 857.0 831.0 815.1 7 Phe
684.1546.8 476.2 362.8 668.0 530.8 460.2 6 5 4 3 2 1 His - A l a - Leu Ser - Clu - Lye 6 7 8 9 1 0 1 1 1 2 736.2 873.2 944.3 1057.3 1114.31273.5 764.4 901.3 972.3 1085.4 1172.6 1101.8 1015.1
-
-
-
146.7 1
Lys 3
9596
Amino Acid Sequence of Thioredoxin fromRabbit Bone Marrow Table V: CID mass spectral data for selected peptides resultin9 from S . a w e u s (strain v8) protease digestion Of HPLC tryptic fraction 8. The m/z values of ions f o r particular ion series (see Scheme I) are listed by rows; Cys' indicates carbaxymethylated cysteine. =
( M t H I +
Y Ztl
5 Ser 1
-
4
Ala -
276.0 147.9 258.9 259.9 2 1
Phe
Gln
3
2
b
159.0
= +
423.1 406.1 407.1 3
-
-
G1U
4 406.1 434.1
306.1
5
I
Z+l 1
7
Pie 1
A m 2
b
261.8
606.1 589.9 5
704.3 6
Asn
-
3 347.9 375.3
507.2 4
260.9
408.0 3
-
Val
4 447.0 475.0
5 546.2 574.2
6 693.1 721.1
Thr -
808.7 6
516.1 623.1 5
Trp
Cys'
1
5
Phe
2
-
Val -
1
-
I10
Glu
7 806.0 834.2
8
I n i H ) ' = 1 215.1 Y 10
Phe 1
Sex'
-
8
Ala 3
2 234.8
b (ntn)+
9
-
7
6
-
461.8 4
404.8 3
307.8 147.0 1 2
Gly
Pro
Cys'
7
-
8
-
9
-
1
Lys 0
=
1171.4 1256.2
813.0 915.4
Y 985.3 Ztl
13
Val 1
b d
1214.4 12 Le" 2 184.8 212.8
-
1
Asp
-
3
1
1
Ser 4
0
9
- Ala - Gly 5
327.8
6
-
8
Asp
-
7
656.2 7 6
-
Lys
Leu -
8
658.1
5
4
Val -
3
Val -
2 Val
1
- Asp
9 1 0 1 1 1 2 1 871.5 970.4 1069.1 1168.1 899.2 998.3 1097.6 1196.2 829.2 956.1 1055.3 1154.2
786.1
3
Table IX: CID mass spectral data for Selected w-chymotryptic peptides. The m/z Valuee Of ions for particular ion series (nee Scheme I) are listed by rows.
Table XI: CID inass spectral data of selected tryptic/themolytic peptides R / Z values of ions for particular ion series (see Scheme I ) are listed by rows: Cys* indicates carbaxymethylated cysteine.
m+ =w
"
-
Y
566.3
Ztl 6
Val
-
1
b
W'
Val 1
b + -
=
467.3 451.1 4
368.3
253.3
3
2
1
-
Val -
Asp -
Phe
2 171.4 199.3
3 270.4 298.4
4 385.4 413.4
5 532.3 560.3
833.2 807.2 791.2 7
762.2 716.1 720.3 6
Ala
Ala
Ser 6
-
m+
=
Y 5
5
Val -
-
419.0 4
380.0 3
Val
Val
-
-
2 3 170.9 270.0 298.0 198.9
280.9 2 Asp 4
-
165.3 1
Y
Phe
Ztl
5
8
Ile 413.0
-
1
b
978.1
-
2 157.3 185.2
-
3 256.1
590.4 429.2 691.2 562.1 665.2
5 G1U 4 357.4 385.2
300.3
536.3 4
-
CYS' 5 518.4 546.1
315.3 359.3 3 Glu 6 641.3 675.1
-
272.3 246.2
173.2 147.3
2
1
Val -
Lys
7 746.5 774.0
8
749.2
x Y Zll 8
Phe 1
b d
857.4 811.2 815.2 7
694.3 678.4 6
xis
Ala -
-
2 251.1 285.1
3 356.2
564.3 649.1 623.3 510.2 607.4 494.2 5 4 Leu - SeP 4 5 441.3 528.3 469.2 556.2 399.1
-
294.2 166.1
Y
1
3
Glu
-
6 657.3 685.3 599.3
2
1
Lys -
Phe
7 785.4 813.5 728.3
8
Ile 1
b
966.4 752.5 653.1 735.5 0 9 8 7 6 5 G1U - Val Asp - Val - Asp 2 3 4 5 6 7 215.3 314.3 429.3 528.3 643.3 243.3 342.2 457.3 556.5 611.4
-
-
1
Asp
-
421.0 406.1 3
Cys*
8
9
786.3 947.5
-
2
Lyr
-
1 1075.2
Scheme I Notation Of Fragment Ions1
Table X : Amino acid analysis of the latest eluting --chymotryptic HPLC fraction.
ser GlY l i b
Asn/Asp LY 5 Glu/Gln Le"
Ile Thr
1.04 1. I4 2.00 2.06 1.76 3.51 2.85 0.89 0.98
1 - 1 CHR H-(NH-CHR-CO)..,-NH-CH
d,
$HR CH-CO-(NH-CHR-CO)..~-OH
W"
'Internal Pragment ions are indicated by their single letter code, e.g., NT has the structure: H-Nl-cmN-co-nn-~v-co*. R R etc. represent the side chains of the amino acii'at2iositibns 1, 2, etc. R is the beta substituent of the nth amino acid.
1
Asp 0
rhe
Amino Acid Sequenceof Thioredoxin from Rabbit Bone Marrow Table VII: CID 185% Spectral data far selected S . aureus (strain v8) protease peptides. The m/z values of ions f a r particular i o n series (see Scheme I ) are listed by rows: Cy** indicates carbaxylaethylated cysteine.
"= 796.1 Y Zil
423.2494.3709.3 692.4 693.3 7 6 Ser - L y s 1 72 1
-
b d
5
Ser 3
,
216.2
4 Ala 4. = 346.2 374.3
-
d
303.2
406.2
276.1 259.2
3 Phe "5 .
2 Gln 6L
-
1 Glu ?7
-
493.4
621.3
521.3
649.3 564.3
(MIHI- = 1 0 1 1 . )
421.3 Y 8 Val 1
921.4 7 Lys
-
2
L'
784.4 6 Cys* -
-
3
228.2
623.3 5 Met -
492.3 4 Pro
-
4
294.3 2 Phe -
690.4 718.2
837.3 865.3
5
492.3 520.2
389.3
-
3
Thr 6
589.3
7
147.2 1 Gln 1
[ntH1- =
632.2
Y 9 LY 5
-
Phe
7 ASn 3 362.2 390.1 319.2
-
248.2 216.2
b d
942.3 1001.4 975.5
Y Ztl
1 Phe 1
-
b d
648.5
=
tn+n,+
-
476.1 504.2 433.3
-
8
-
2 235.1
292.2
363.1
799.3 899.5 873.4 857.4
671.2 771.2 745.3 728.3 729.5 7 LYS 4
1
6
674.2 702.3 660.3
572.2
760.3
646.4389.3 518.2 629.3 630.4 502.4 5 1 L y s - Glu 7 8 706. 734.3
6 As"
5
6
1046.3 1020.6 1004.3
Ztl
-
9 Phe 2
-
8
Lys
-
3
617.4 601.4 6
GlY 5 608.5
295.3
b
d =
L H t H ) +
-
b d =
GI" 9
443.1 372.2 3 Lys 9 4 862.5
2
Leu 1 947.4 975.5 905.4
486.3 586.4 560.1
432.1
205.2
543.0 5 Gln 6 708.2 736.5 651.4
4 Lys 7 836.2 864.5 779.2
2 3 Vcil - Gly 9 8 935.5 963.5 1020.6 921.5
948.2
1277.4
U'
1
-
11 7 Asp 2 187.3 215.2
-
1136.4 10 8 Val 3
-
314.2
9 Asp 4 10
Asp 5 9
Cy=* 68
429.3
544.1
705.0
-
646.4 6 Lys 7 805.3 832.9
544.1
429.3
5 Asp
lie -
4
-
1567.8 1426.6
962.1 936.4
1097.1
1524.5 8
920.3 14 10 Val 1
1311 Asp 2 187.3
-
215.3 b
Y
808.1
at1
7 ASP a b
693.3 677.2 6
12
Val 3
Asp 4
-
314.3
429.3
606.3 580.3
509.1
Asp 5
-
544.4
-
Ile
-
-
1986.5
Y
1432.8 18
17 Lyn 2
-
1
-
16
Cys' 3
-
Cy$* 6 677.2 705.3
Lys 7
-
833.2
309.1
5 4 3 Ala Ala G1U 8 9 10 11 12 920.3 1033.5 1104.5 948.5 1061.8 1132.7 1203.8 1332.7
Val
-
-
2 Cy=* 13
-
1
Glu 14
1695.1 1598.0 1221.6 1798.4 1468.6 1192.9 1771.5 1641.4 1544.0 1412.8 1295.3 1167.5 1755.1 1526.7 1425.7 1279.4 1150.9 15 14 13 12 11 10 Met Pro - Thr - Phe Gln - Phe -
-
l
-
i
L
.
-
?
836.9 520.1 389.3 228.2b
616.9 882.2
x
Y
1045.9 1020.0
-
a b C
d
1 G1U 10
*"
.
I
1112.3 993.7 1140.8 1156.9
329.9 873.4
745.1 728.1
616.9
-
432.3
-
Lys - Lys - Gly Gln L y s - Val -Phe 10 11 12 13 I4 15 16 1238.8 1387.3 1516.1 1700.5 1829.0 1928.5 1286.9 1415.5 1544.0 1600.4 1729.0 1855.8 1955.9 1304.6 1432.1 , 6 1 7 ..1 17dF. 7 ~~. 1329.9 1458.6 1643.8 1771.5
290.3 3 Ala -
2 Ala 11
-
920.1 1033.2 948.2 1061.1 1132.3 1203.5 1005.2
1640.3
Y Ztl 9
1 G1U 0
1350.3
Y 12 Val 1
261.2 2 11.3 8 934.2 962. II 906.6
3 Phe 7 821.1 849.3
-
700.3
7
Ala
Gly 3
4
Val
1167.3
Y 10 Phe 1
5 Val 5 575.3 603.2 561.3
-
914.4 888.3
959.3 0 9
Ser -
Asn
G l y . Glu 17 18
1 G1U 12
9597
