2621
Anal. Chem. 1987, 59, 2621-2625
Novel Fragmentation Process of Peptides by Collision- Induced Decomposition in a Tandem Mass Spectrometer: Differentiation of Leucine and Isoleucine Richard S. Johnson, Stephen A. Martin,' and Klaus Biemann* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
John T. Stults and J. Throck Watson Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
The mass spectra produced upon collision-induced decomposition of the protonated molecules of peptides often exhlbit peaks that correspond to Ions that are formed by cleavage of the -N-CR- bond along the peptlde chain followed by cleavage of the &-y bond H R has the general structure -+Cy-R'. Ions produced In this manner are assigned the notatlon w, and are helpful In the characterlzatlon of the amino acid at that positlon. Most important Is the dtfferentiatlon of the amlno acids leucine and Isoleucine, whlch Is generally ditflcuit or hnposslMe by mass spectrometry. Aromatlc amino acids do not undergo thls fragmentation because It would Involve cleavage of a C-Ar bond, neither does alanlne, whkh would Involve loss of a hydrogen radical, nor does giyclne, which lacks a B,-y bond. The nature of thls fragmentatlon process Is demonstrated by exact mass measurements and precursor-product Ion studies.
The collision-induced decomposition (CID)mass spectra of peptides are very well suited for the determination of their amino acid sequence. In contrast to the normal fast atom bombardment (FAB) spectra (I),they exhibit extensive series of the same ion type. The most frequently occurring are ions of type an and b,, generally observed if a basic amino acid is located at or near the N-terminus or if there is no basic amino acid, and y, if one is located at the C-terminw. Often all three ion series are present, with one or two of them dominating (2). This is in contrast to normal FAB mass spectra, which rarely exhibit continuous series of ions of one type and are difficult to interpret in the low-mass region, which is obliterated by the contributions of the matrix. One way to improve the signal-to-background ratio is to use a very high concentration of pure peptide in the matrix, a situation that rarely prevails when working with peptides of unknown sequence derived by proteolytic cleavage of a protein of unknown structure. The generalized structures of fragments produced upon FAB from peptides of the typical structure I are shown in Table I. Detailed fragmentation processes leading to these ions have been suggested previously and have been summarized in a recent review (3). The nomenclature used here for the various ion types is a variation of that proposed by Roepstorff and Fohlman ( 4 ) . In the course of our work on peptide sequencing with high-performance tandem mass spectrometry, which permits the selection of the 'q species of a (M + H)+isotopic cluster On leave of absence from the Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC.
Table I. Fragments Produced from Protonated Linear Peptides (I) R R R H,N-cH-CO-(~H-CH-CO),-NH-LH-COOH I
+H
I+
I
in MS-1and the determination of its CID spectrum in MS-2 with an accuracy of better than Et0.3 u (5), we have often observed a peak which occurred 54 mass units higher than that corresponding to an ion of type y. This mass increment implied the retention of a remnant of the next N-terminal amino acid and would nominally correspond to the loss of the R group plus two hydrogens from the next y ion (y,) as shown in path A.
Yn
Vn.i+54
These fragment ions were assigned the notation w, (6). However, as CID spectral data of a large number of peptides of known sequence (7) as well as those derived from proteins of unknown sequence accumulated, it became clear that this fragmentation process is much more dependent on the nature of the N-terminal amino acid of the fragment ion than is the case with the other sequence-specific ions (an, b,, c,, x,, yn, z,,), which involve only the cleavage of a bond along the peptide backbone, sometimes accompanied by the rearrangement of a hydrogen atom (Table I). Most notable was the absence of w ions when aromatic amino acids, glycine, or alanine were involved or those that carry a substituent on the p carbon, such as valine, isoleucine, and threonine. For the latter three, ions were observed at higher mass corresponding to the retention of one of the substituents at the p carbon. This observation suggested that the formation of the w, ions involves the cleavage of the p,r bond because such a fragmentation would be difficult for aromatic amino acids where it would require cleavage of a CH2-Ar bond. It also would cause
0003-2700/87/0359-2621$01.50/00 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987
Table 11. Exact Mass Measurements peptide"
no. in Table IV
path A elem comp of w, ion
calcd mass
RPPGFSPFRb LQQZGALK LQQIGALK
13
C22H31N705
413.2387
(I
17 17
path B or C elem comp of w, ion calcd mass C23H33N605 CZ1H38N506
C25H45N707
555.3381
C26H47N607
measd mass
473.2513 456.2822 555.3506
473.2510 456.2841 555.3510
Amino acids in single letter code: for three letter code see Table 111. w, ion at italicized amino acid.
retention of the substituents at the 0carbon, but not at the N-terminal nitrogen atom. Furthermore, path A was ruled out on the basis of exact mass measurements in the normal FAB mass spectrum of two peptides that exhibited w, ions of sufficient intensities (Table 11).
EXPERIMENTAL SECTION The CID mass spectra were determined either with a two-sector magnetic deflection mass spectrometer (the JEOL HXllO at MSU) operated in the linked-scan mode and using the collision cell in the first field-free region (after the ion source) or in a four-sector tandem mass spectrometer (the JEOL HXllO/HX110 at MIT) with the collision cell in the third field-free region (5). Two-Sector Linked Scans. A 1-pL aliquot of the peptide solution (1-3 nmol/pL) was mixed with 1 pL of matrix (either glycerol or 5:l dithiothreitol/dithioerythritol)on the FAB probe tip. The precursor ions were formed by fast atom bombardment with a 6-keV Xeo beam in a JEOL HXllO mass spectrometer (EB geometry) operated at 10-kV accelerating voltage, resolving power = 3000. Helium was admitted to the collision cell (first field-free region) to the pressure required to attenuate the precursor ion to 30% of its original abundance. The JEOL DA5000 data system generated the linked scan a t constant B / E ratio. The raw data profiles were acquired (30 s/decade) with the accumulation software, summing 10-15 spectra/sample. Two of the peptides used (Table 111, no. 10 and 12) were commercially available. Other peptides were from trypsin digests of proteins (Table 111: no. 8, rubisco from spinach; no. 11 and 17, thioltransferase from rat liver; no. 23, Hzofrom tobacco) for which the sequences are known. Four-Sector Tandem Mass Spectra. To a solution of 1-2 nmol/pL in 30% aqueous acetic acid was added an equal volume of glycerol and 1pL of this mixture was placed on the FAB target, which was then inserted into the ion source of the JEOL HXllO/HX110 spectrometer. Ionization was achieved by bombardment with a Xeo beam of 6 keV kinetic energy. The accelerating voltage in MS-1 was 10 keV. The "C species of the (M H)' isotopic ion cluster was selected by MS-1 and this precursor ion beam was transmitted through the collision cell into MS-2. Helium was admitted into the cell at such a pressure (approximately Torr) that the precursor ion was attenuated to about 30% of its original abundance (as measured at detector 3, after the collector slit of MS-2). The CID mass spectrum was recorded by the JEOL DA5000 data system which also generated the B/E linked scan of MS-2. The spectrum shown in Figure 2 is from a single scan recorded in 1.9 min and represents the raw data profile. To record the product ion spectra (Figure 1)of fragment ions produced in MS-1, the latter was set to transmit these fragment ions (one after the other) into the collision cell. In each case, the product ion spectra were recorded in the same manner as outlined above for (M + H)' ions. The peptide samples were either commercially available (Table 111: no. 4, 5, 9, 13-15, 18, 22, 25-28, and 30-33) or generated by proteolytic digestion of thioredoxins from E. coli and from Anabaena, small proteins of known structure (9,lO) (Table 111: no. 1-3, 6, 7 , 16, 19-21, 24, and 29). Mass Measurements. Exact mass measurements for ions in the normal FAB spectrum were made with a JEOL HXllO mass spectrometer at a resolving power of 10000 in the peak-matching mode. Glycerol cluster ions were used as reference masses.
+
RESULTS AND DISCUSSION Two pathways (B or C) could account for the formation of such a fragment ion from either a z, or a z, + 1 ion as outlined
Table 111. Partial Listing of Peptides Used in This Study with the Amino Acids that Give Rise to w, Ions Underlined 1. Tyr-Gly-&-Arg 2. Gly-Gln-Leu-Lys
3. Leu-Thr-Val-Ala-Lys 4.
