1891
Anal. Chern. 1990, 62, 1691-1695
Differentiation of Hydroxyproline Isomers and Isobars in Peptides by Tandem Mass Spectrometry Daniel B. Kassel and Klaus Biemann*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
The lsomerlc 3- and 4-hydroxyprollnes are Isobaric with the Isomers leuclne and lsoleuclne, and all four have, therefore, the same “residue mass” of 113. Secondary fragmentatlon processes were found that dlfferentlate the hydroxyproline Isomers from each other and from the leuclnes. Variants of synthetic bradykinin containing one or two hydroxyprohe moletles were prepared by using manual Edman degradation and/or enrymatlc methods. The tandem mass spectra of these peptides were recorded. The C-termlnal w, fragment ions allow the dlfferentlatlon of 4-hydroxyproline from the 3-Isomer and lsoleuclne, while the N-termlnai a, Ions contalnlng 4hydroxyprollne undergo H,O ellmlnatlon to differentiate thls amino acld from the 3-Isomer and leucine. Lys-C digestion of a mussel adheslve protein produced a set of decapeptldes varylng In the degree of hydroxylatlonof proline and tyrosine. Heterogeneity with respect to 3-hydroxyprollne and 4-hydroxyprollne at a certaln posltlon in these peptides was assessed by tandem mass spectrometry based on the w, ion serles In the C I D spectra of these Lys-C peptldes. Some N-termlnal Ions further allow for the dlfferentlation of these two Isomeric species.
INTRODUCTION Hydroxyproline (Hyp) is a cyclic amino acid formed by the hydroxylation of proline during the posttranslational processing of proteins by a prolyl hydroxylase (1,2). The presence of Hyp-containing peptides in mammalian cells is associated with connective tissues (e.g., collagen and basement membrane collagen) (3-5). The principal form in collagen is the trans-4-L i~omer(~Hyp), but in type IV (basement membrane) collagen, the trans-3-L isomer (3Hyp)becomes more prominent. Recently, several Hyp-rich peptides have been reported in human urine and plasma, most notably [Hyp3]-bradykinin, which is attributed principally to high molecular weight kininogens and in some cases to end products of collagen catabolism (6-8). This bradykinin analogue has been found to be hydroxylated in either the 3- or 4-position. In addition to collagen, Hyp-rich proteins have been found in a variety of plant species. The occurrence of hydroxyproline in these plant proteins is known to play a role in growth regulation and provide the structural component of primary cell walls. Primarily associated with extensin, these proteins are hydroxylated exclusively at C-4 of proline, about half of which are 0-glycosylated (9,10). In addition, hydroxyproline has been observed in a variety of marine organisms that produce highly hydroxylated polyphenolic proteins to provide the adhesive properties necessary to resist the buoyant effect of sea water (11-13). These proteins consist of repeating decapeptide units (more than 80/molecule), which exhibit a high degree of heterogeneity with respect to the extent and position of hydroxylation of three prolines and two tyrosines. *To whom correspondence should be addressed at Department of Chemistry, Room 56-010, MIT, Cambridge, MA 02139. 0003-2700/90/0362-i691$02.50/0
- -
Scheme I. General Fragmentation for the Formation of d, and w, Ions H+
H+
H-(NHCHRCO),-l-NH
--H*
+
H-(NHCHRCO),-I-NH
-EH PZH,
a,,
+1 H+
0
m
ii
*CH-C-(NHCHRCO),,-1-OH P I
H+
2-
II
* CH-C-(NHCHRCO),-I-OH
A. 2, i1
Tandem mass spectrometry utilizing collision-induceddecomposition (CID) has proven to be a reliable method for sequence determination of proteins and peptides (14-16) and is complementary to the gas-phase Edman sequencing technique. The protonated peptide (or precursor ion) of interest (produced by fast atom or Cs+ ion bombardment) is individually mass selected by using the first mass spectrometer (MS-1) and subsequently transmitted into a collision cell region where it undergoes CID with a neutral gas (e.g., helium). The fragment ions formed upon CID are then separated and mass analyzed by using the second mass spectrometer (MS-2). We now have a good understanding of the fragmentation of protonated peptide molecules containing the 20 common protein amino acids (17-19). It remains to investigate peptides containing hydroxyproline, a rare amino acid occurring only in certain types of proteins (see above) where it is produced by posttranslational hydroxylation of proline. Hydroxyproline has the same residue mass (113 amu) as leucine and isoleucine, and the normal sequence ions (a,, b,, c,, x,, y,, and z,) will therefore not differentiate these three but will of course allow for the differentiation of Hyp and Pro. It has been demonstrated previously in this laboratory that the side-chain sequence ions (i.e., d, and w,) are useful for differentiating isoleucine from leucine (17, 18). The genesis of these two ions is shown in Scheme I. Isomeric amino acids differing in the substitution of the @-carbontherefore generate d, and w, ions that differ in mass correspondingly and consequently permit one to distinguish leucine from isoleucine. A large body of data generated in this laboratory has demonstrated that a, ions are accompanied by d, ions, both of which are derived from a, + 1 ions (18), if there is a basic amino acid at or near the N-terminus and the nth amino acid has an easily cleaved @,?-bondbut is not cyclic. Thus, a CID spectrum that exhibits dominant d, ions but lacks such a peak for an amino acid of residue mass 113 should indicate that the peptide contains a hydroxyproline, rather than leucine or isoleucine, at that position. Both Pro and Hyp do produce w, ions when a basic amino acid is present near or at the C-terminus of the peptide. The m / t value of a w, ion for 4Hyp should permit the differen0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
Table I. Peptides Utilized in Studying Hydroxyproline Fragmentation Behaviornnb I
I1
I11
RPPGFSPFR RPPGFSPF PPGFSPFR PPGFSPF
RPPhGFSPFR RPPhGFSPF PPkGFSPFR PPiGFSPF
RPPGFSFFR RPPGFSFF PPGFSFFR PPGFSFF
IV
RRPhPhGFSFFR RRPhPhGFSFF RPhPhGFSFFR RPiPiGFSFF e PhPhGFSFFR f PhPhGFSFF a I, bradykinin; 11, [Hyp3]bradykinin;111, [~-Phe?] bradykinin; IV, [D-Arg, Hyp2v3,~-Phe']bradykinin. (a) Unmodified peptide; (b) carboxypeptidase B digested; (c) manual Edman ( X l ) degraded; (d) manual Edman (Xl)and carboxypeptidaseB digested; (e) manual Edman ( X 2 ) degraded: (0 manual Edman ( X 2 ) and carboxypeptidase B dia b c d
Table 11. Peptide Sequences from Mussel Adhesive Protein (MAP) by Endoproteinase Lys-C Digestion m / z [M + HI+,
peptide sequenceb
amtc
707
A-K-P-T-Y-K A-K-P-T-Yh-K Ph-S-Y-Ph-Ph-T-Yh-K A-K-Ph-S-Y-Ph-Ph-T-Yh-K
0.25
723 1016 1215 1231
0.50 0.50
0.25 0.75
A-K-Ph-S-Yh-Ph-Ph-T-Yh-K
a Determined by FAB-MS. *Determined by tandem mass spectrometry; Ph hydroxyproline; Yh = dihydroxyphenylalanine (Dopa). Nanomoles (estimated from OD,,,).
gested. *,-L
tiation from Ile but not Leu (which would have a w, ion of same mass). Furthermore, the presence of w, ions should allow differentiation of the 3Hyp and *Hyp isomers: The w, ion of 3Hypwould be 16 amu heavier than the corresponding w, ion for 4Hyp because the former fragment retains the OH group. Although 3Hyp produces a w, of the same m / z as threonine, this presents no particular problem since threonine has an altogether different residue mass. It therefore remains to be determined whether 4Hyp can be differentiated from Leu on the basis of the formation of some specific additional fragmentation, such as the loss of HzO. In an effort to support these expectations, the CID fragmentation of a number of synthetic peptides containing Hyp has been investigated. Results are presented that corroborate the behavior predicted above. The results of this study were then used to determine the degree and position of proline hydroxylation in Hyp-containing peptides from tissue extracts of the adhesive protein of the mussel, Mytilus edulis L. The results demonstrate the utility of tandem mass spectrometry for the differentiation of isomers of hydroxyproline. EXPERIMENTAL SECTION Reagents: Phenyl isothiocyanate (PITC) and hexafluoroisopropyl alcohol (HFIP) were purchased from Aldrich Chemical Co. Trifluoroacetic acid (TFA) was obtained from Pierce, Co. Enzymatic digestion and chemical cleavage of bradykinin analogues: Peptides I-IV (see Table I) were purchased from Boehringer-Mannheim. Manual Edman degradation was carried out on ca. 10-20 nmol of peptide with 20 pL of 5:19:1 PITC/ pyridine/HFIP at 50 "C for 10 min (20). Reactions were terminated by addition of 10 p L of TFA and subsequent lyophilization. Removal of C-terminal amino acids was achieved by digestion with carboxypeptidase B (Boehringer-Mannheim)at 37 "C for 12 h and an enzyme/substrate ratio of 1:lOO. An aliquot (1110) of the reaction product was separated by reversed-phase HPLC using a C4or CIS4.6 mm X 250 mm column (Vydac, Inc.). The solvent gradient was from 5% to 65% acetonitrile in 0.035% aqueous TFA over 30 min. Glycerol (2 pL) was added to collected HPLC fractions prior to speed-vac lyophilization (Savant, Inc.). Digestion of mussel adhesive protein (MAP): Purified MAP, provided by Biopolymers, Inc. was digested with endoproteinase Lys-C (Boehringer Mannheim) analogous to the tryptic digest of Waite and co-workers ( 1 1 ) . Approximately 2 nmol of purified protein was reacted at an enzyme/substrate ratio of 1:1OO, in 200 pL of 0.1 M sodium borate adjusted to pH 8.5 at room temperature for 12 h under NP. The reaction was terminated by lyophilization and subsequently dissolved in 0.5 mL of 0.1% aqueous TFA. Similar to the results of Waite et al., the digest contained two hexapeptides and a number of decapeptides (see Table 11). These peptides were partially fractionated by HPLC with a solvent gradient of 5%-40% acetonitrile in 0.035% aqueous TFA over 35 min after an isocratic period of 5 min at 5% acetonitrile in TFA. Tandem mass spectrometry: To each HPLC fraction was added 2 pL of glycerol prior to speed-vac lyophilization, and the resulting material introduced into the ion source of a JEOL HXllO/HX110 tandem mass spectrometer. The sample was
I
t
2-r
30
53"
4 7
-C"
8
7
Figure 1. CID mass spectrum of (a) IC and (b) IIc (from Table I). A w, ion series is present, and the w, ion is the same for both peptkies: secondary electron multiplier (SEM) = 1 4 keV
ionized by using a JEOL cesium gun operated at 25 keV and 2.80-A heating current. Mass spectra were obtained by scanning the magnet of MS-1 from 300-3000 amu in 90 s. MS-1 resolution was 1:1600,and the electron multipliervoltage was 1kV. Collision spectra of peptides were obtained by operating the instrument in the MS/MS mode, and the resolution of MS-1 was such as to select the lZCisotope component of the (M + H)+ion. Helium was used as the collision gas in the field-free region between MS-1 and MS-2 at a pressure at which the precursor ion current was reduced to approximately 30% of its original intensity. Collision spectra at 7-keV collision energy were obtained by scanning Bz and E2at the appropriate ratios, from m/z 30 up to the precursor ion. The electron multiplier was typically operated at 1.4-1.9 kV. The resolution of MS-2 was set to 1000 (dynamic). RESULTS AND DISCUSSION To investigate the fragmentation of hydroxyproline-containing peptides, the CID spectra of a series of bradykinin variants (Table I) was recorded. Shortened variants of bradykinin (Ia), [Hyp3]bradykinin (IIa), [~-Phe']bradykinin (IIIa), and [~-Arg,Hyp~p~,~-Phe~]bradykinin (IVa) were prepared by one or two Edman steps and/or enzymatic cleavage of C-terminal residues with carboxypeptidase B. For those bradykinin variants containing an Arg moiety at the C-terminus, only the C-terminal ions (Le., x,, y,, z,, w,, v,) are observed in their CID spectra, as predicted. Shown in Figure 1 are the CID spectra of [des-Arg'lbradykinin (IC) and
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 x4 0
r
(a)
' 1
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
I?
Scheme 11. 1,2-Elimination of Water from the a, Ion of 4-Hydroxyproline
05
H
i 13
1693
H i
i
a,- H20
AH
a,
+1
an
Scheme 111. Influence of Adjacent Hyp Residues on the Fragmentation of the all,. 1 Fragment Ion
+
I
Arg-Pro-Hyu-Glv-Phe-Ser-PIO-Phe . .. .
1
p:
ady 605
7c0
53:
+ 1 (&z
5
397)
%ly
+ 1 -OH
(&z 380)
9'33
Figure 2. CID spectrum of (a) I b and (b) Ilb. The region of potential d, ions for leucine and isoleucine (arrows) is expanded; SEM = 1.4 keV.
[Hyp3,des-Arg']bradykinin (IIc) produced upon a one-step digestion of [Hyp3]bradykinin (IIa). The w, ion series predominates and is interrupted only by the v, ions that are thermodynamically favored for phenylalanine and tyrosine residues (18), shown as v2 and v5. The wl ion at mlz 764 corresponds to the 4Hyp residue in this bradykinin variant. The w7 ion due to fragmentation at Pro or 4Hyp appears at the same mass (mlz 764) in both species. This is a consequence of the fact that the w, ions for 4Hyp do not retain the hydroxyl moiety and are therefore identical with the w, ions for proline. However, this presents no problem for differentiating the two amino acids because they differ in mass by 16 m u . Two possible amino acid sequences that can be derived from the CID spectrum in Figure l b are the following:
Pro-(kT]-Gly-Phe-Ser-Pro-Phe-Arg The ambiguity in assigning position 2 as either leucine or hydroxyproline is due to both amino acids giving rise to wl ions of identical mass (and structure). Isoleucine may be eliminated since the w7 ion would be observed at mlz 778, instead of mlz 764. To make this distinction, we usually convert the peptide to a derivative with a fixed positive charge at the N-terminus (21,ZZ). However, in this case the analogous peptide with a basic Arg amino acid at the N-terminus rather than the C-terminus was easily prepared. A comparison of the CID spectra of [des-Argg]bradykinin (Ib) and [Hyp3,des-Ar2]bradykinin(1%) in Figure 2 shows ions unique to hydroxyproline that differentiate this amino acid from the LeuJIle isobars. In the CID spectrum of IIb, the a, and d, ion series are predominant due to the presence of a basic moiety at the N-terminus of the peptide. Both spectra show significant d5 and d6 ions for Phe5 and Ser6,the only amino acids in this peptide capable of forming d, ions (18). Their intensities are in agreement with our experience that the d,:a, abundance ratio is low (
