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Double stranded DNA sequencing by tandem mass spectrometry
EI,S EVI E R
International Journal of Mass Spectrometry and Ion Processes 165/166 (1997) 457-466
andIonProcesses
Double stranded DNA sequencing by tandem mass spectrometry Fred W. McLafferty”, David J. Aaserud, Ziqiang Guan, Daniel P. Little, Neil L. Kelleher Baker Chemistry Laboratory, Cornell Uniuersity, Ithaca, NY 14853-1301, USA
Received 28 April 1997; accepted 16 June 1997
Abstract Electrospray ionization produces far more abundant molecular ions for double stranded (ds) DNA than for single stranded (ss) and accurate molecular masses can provide the base composition of dsDNA. This study shows that the Fourier-transform mass spectra of a ds 64-mer DNA can provide approximately 50% more sequence information than the spectra of either individual ss component. Cleavages triggered by T loss are of low probability, but the opposite is true in the complementary ss strand that bears the base A at this site. The spectrum of the product of the attempted biological synthesis of a ds 70-mer DNA provided critical information on its extensive 3’-end heterogeneity. Published by Elsevier Science B.V. Keywords:
Electrospray;
Fourier-transform
MS; DNA; Tandem; Sequencing
1. Dedication It is a special privilege to contribute to an issue honoring Professor Keith Jennings, one of the true pioneers in molecular mass spectrometry.
*Corresponding author. Tel.: + 1 607 2554699;fax: + 1 607 2557880. 0168-3659/97/$17.00 Published by Elsevier Science B.V. PI1 SO168-1176(97)00171-7
For example, his prescient paper 3 decades ago demonstrated the collisionally activated dissociation (CAD) of benzene ions, while a very recent paper concerns CAD of the much larger biomolecule, bradykinin [ll. Hopefully, this paper on CAD of a 39-kDa double stranded DNA offers a useful extension of the unusual professional contributions of Keith Jennings to mass spectrometry.
458
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et al. /International Journal of Mass Spectrometly and Ion Processes 165 / 166 (1997) 457-466
2. Introduction
ton dissociation (IRMPD) of the molecular ions. For the a, and w, ions of n = l-10, four were not found, of which three would have required T loss; for II = 11-20, 13 were not found, six with T loss; and for II = 21-30, 14 were not found, four with T loss. Because the most easily lost base, A, is the Watson-Crick complement of the least easily lost, T, backbone cleavages not represented in the ESI/FTMS spectrum of a single stranded (ss) DNA should have a relatively high probability of being represented in the spectrum of its ss complement. This is examined here with spectra of the ds DNA and its 64-mer ss complements used in the base composition study [19].
Sequencing of linear biomolecules by tandem mass spectrometry (MS/MS”) is well accepted. Its unique specificity and sensitivity are extensively documented for peptides [2-51 and larger proteins [6-91. Only recently, MS” sequencing has been extended to nucleotides (> lo-mers) [lo-141, made possible by the special advantages of gentle electrospray ionization (ES0 [15] and the high resolving power (> lo51 [6] of Fouriertransform @T) MS” 1161. ESI/FTMS has provided the complete sequence of a 50-mer DNA and extensive sequence data for DNA as large as a 108-mer [ill. Recent studies employing double-stranded (ds) DNA [14,17-191 show that ES1 yields abundant molecular ions of it and its two single strands (ss), whose exact masses can yield the base compositions of the ds and its ss components [19]. This derivation takes advantage of the Watson-Crick base complementarity of the two ss components (A and C in ss1 paired with T and G, respectively, in ssI1 and vice-versa). Here this complementary information is used to provide additional sequencing data for larger DNA molecules. The previous ESI/FTMS study of nine 42-108mer DNAs [ill showed that the most definitive sequence information comes from the terminal fragment ions. The 5, ions that contain the 5’terminus and the wn ions that contain the 3’terminus can be as large as 10 kDa to provide partial sequence information for up to approximately 30 bases from each end of the DNA molecule.’ The backbone cleavages forming the g, w and internal (i> ions are triggered by loss of an adjacent base, with base A loss favored [lO,ll]; however, base T loss is not found except in unusual cases (e.g. adjacent T bases) ill], so that the corresponding sequence fragment ion is not formed. This problem can be illustrated by a 50-mer DNA [ill for which the complete sequence could be derived from infrared multipho-
4.1. ds 64-mer
‘These a, ions were originally designated as a, +, - Base [9]; thus for gn, w,, and j, (internal ions), small II is the number of bases in the ion [IO].
ES1 ions from the annealed complementary 64-mers were trapped in the FTMS and subjected to IR laser [201 or BIRD 121,221heating to obtain molecular ions (M-1. These were abundant for
3. Experimental Procedures generally follow those reported recently [11,19-211. The complementary 64-mer DNAs and the 40-mer DNAs complementary in their first 10 bases (5’ vs. 3’) were synthesized and HPLC desalted at the Cornell Peptide/DNA Synthesis Facility and annealed for 5 min at 95°C. The ds 40-mers were then extended with Klenow fragment (a section of DNA polymerase I). Solutions were prepared as 5 PM (CH,CN-H,O, 3:l) with 0.02% piperidine and electrosprayed (- 2.5 kV, coaxial SF,) at 1 pl/min. The modified 6 T Finnigan FTMS instrument (Odyssey data system) consists of an external injection ES1 source with three rf-only quadrupoles for ion transportation into the trapped ion cell. Typically, ions were collected with the source/analyzer trap plates at -3 and -4 V and a N, pressure pulse (approx. 1O-6 torr) for enhanced trapping. For blackbody infrared radiative uissociation (BIRD) experiments [21,221, the traps were held at -2.5 V during the delay and reduced to - 1 V prior to broadband excitation and detection. 4. Results and discussion
F. W. McLaffeq
et al. /International Journal of Mass Spectromety and Ion Processes 165 / 166 (1997) 457-466
Fig. 1. Effect of IR laser irradiation time on ion yields from the ds 64-mer
both the double and single stranded (ds, ss> components whose mass values defined the base compositions of both ss DNA molecules [19]. The Mincrease with initial laser heating (Fig. 1) presumably resulted from ‘boil off of non-covalent adducts [21,23,24]. With increased IR irradiation, the ss M- ions abundances maximize before that of the ds M- ions, consistent with more strongly adducted ds M- ions and/or minimal fragmentation of ds M- ions to produce stable ss M- ions. IRMPD (50 ms, Fig. 2 and 60 ms> [20], BIRD [21] and nozzle-skimmer dissociation [25] spectra of ES1 ions from the ds sample and IRMPD spectra of ions from separate samples of ss1 and ssI1 gave the combined fragment mass data and sequence assignments of Table 1. None of the fragment masses of ds spectra was assignable to non-covalent complexes of fragments from both strands, although not all masses (omitted from Table 1) were structurally assignable. In such ds spectra, the fragments thus arise from either of the ss M- ions; the same interpretation problem was encountered for MS/MS of a mixture of 42 kDa proteins [S]. For the ss DNA mixture, molecular origin is clearly defined for fragment ions whose masses sum to that of one of the ss Mions; the peak masses 8132.29-4 &) plus 11607.92-6 (Wan)plus 135.05 (base AH lost in this fragmentation) sum to 19875.26-9 vs. the measured value 19874.33-9 for M- of SSI [19]. However, those fragment masses also formed from the individual ss DNAs (Table 1) clarify any ambiguity of their molecular parent. MS/MS spectra of
459
the individual ss1 and ssI1 molecular ions could provide similar delineations but were not measured here. Except for peaks of relatively small abundance, accuracies off 0.05 Da are found (Table 1) for most fragment masses up to approximately 2.5 kDa and k 0.2 Da up to 5 kDa without internal mass calibration. As found for ESI/FTMS of the 42 - to 108-mer DNAs [ll], this mass accuracy makes possible confident assignments of base composition (Fig. 3) for most fragments of up to five bases (however, note (Ixy6, A,T,CJ, = 1910.321 vs. (II&, A,T,C, = 1910.335). For higher masses, the accuracy of mass differences is higher, as is the assignment of composition differences. For ss1, a consistent set of 3’-end ions w4,
wg,
wX,
w1O,
wll>
w14,
w15,
w16,
w18,
w19:
w21,
are assignable and w30 (Table 11, with w3, (and _a,, its ss1 complement) assigned above (Fig. 3). Now the structural complementarity of ss1 and ssI1 (5’ to 3’, replacing A with T, C with G and vice versa) can provide further details using the ss-II 5’-end series; u2, a3, aI, a,, a12, al3 and al7 represent cleavages of complementary bonds not cleaved in ss1. The 5’-end ordering ‘C2T for both ss1 and ssI1 is indicated by the absence of an al peak consistent with the unfavorable loss of T [II]. Combining the complementary information for the 39-62 region of ss-I, only 42,43(CG)and 5R,59(AG)in ss-I are not ordered. Using this same approach for the first 16 bases of ss-I, the a ions assign sequences to the first 10 bases, while the w ions of ss-II assign the rest. For the 50-mer DNA [ll], internal j ions provided approximately 35% of the information on the sequence. Base compositions of j ions from spectra containing both ss components will be considered first to find any assignable to a unique position in only one of the ss strands. Thus i(A,CG,) fits only (1127-32, as formation of (11145-50 requires the loss of 44T. The ions i(ACG,) and i(C,G,) must represent (1128-33 and (1129-34, respectively. The ion i(AZCL) fits only (1143-46; both (1117-10 and (1050-53 require T loss. The ion i(A,TC,G) indicates (11)48-54; formation of (1145-51 and (1049-55
w23,
w24,
w25,
w26,
w29
460
F. W. McLaffeny et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466
Table 1 IRMPD (1, 50 ms; 2, 60 ms), BIRD and NS of ssI, ssI1 and ds-64 mer Mass 691.13 771.10 780.11 786.11 796.10 804.12 811.10 820.11 836.11 949.17 1004.19 1020.18 1060.15 1069.15 1084.15 1085.15 1090.14 1093.15 1099.15 1109.16 1115.15 1124.16 1125.15 1133.17 1140.15 1149.17 1238.15 1268.21 1349.23 1373.20 1379.19 1382.21 1388.20 1389.19 1398.21 1404.19 1412.21 1413.21 1419.19 1422.22 1428.21 1429.20 1438.21 1444.20 1453.21 1597.27 1638.28 1662.25 1668.23 1678.24 1686.26 1693.24
‘k”
Assignment
IRMPDssI
(I,II)a2 TC AC
691.11
(T-0 CG A2 TG AG G2
786.10
786.06
1004.15
Wa3 TC2(11)” AC2 ATC C2G T3 A2C AT2 ACG(I)” T2G ATG CG2 A2G TG2 AC2 ATC2-AH(H)
1020.12
(Ihv4 (I)a4 ATCXII) T3C A2C2(1)43-46 AT2C(II)9-12 TC2G01) AC2G(I)” T2CGtII) A2T2 ATCG T3G(1)13-16
1268.19 1349.20
Uhv5 Wa5 ATC3 T3C2 TC3GUI)” A2TC2(11) T2C2GtII)
691.10 771.06
804.10
(10~3 (II)a3
A2CG0) AT2G(II)14-17 TCG2 ACG2 T2G2(11)19-22 ATG2
IRMPDssII
IRMPDlds
771.09 780.10 786.09 796.09 804.10 811.09
949.16 1004.19 1020.18
1060.11
IRMPD2ds
BIRD ds
691.12 771.10 780.10 786.09 796.10 804.10 811.09 820.10 836.10 949.17
691.10
1020.18 1060.14 1069.15 1084.14 1085.15 1090.14 1093.15 1099.16
780.07 786.08 796.08
771.09 780.10 786.10 796.10
811.09
811.09
836.10 949.13 1004.18 1020.20
1099.13 1109.17
1109.16 1115.09 1124.15 1133.17
1268.21 1373.16 1379.16
1115.14 1124.15 1125.15 1133.16 1140.16 1149.15 1238.21 1268.24 1349.23 1373.20 1379.19
1115.13 1124.20 1133.17
1268.19
1398.19 1404.20
1413.15
1413.20
1428.14 1429.15
1429.19
1444.15
1444.00
1419.18
1453.20 1597.24 1638.25
1597.25 1638.28 1662.18 1668.23
1693.17
1268.20
1382.23
1382.25
1404.14
NS ds
1388.21 1389.21 1398.21 1404.20 1412.22 1413.21 1419.19 1422.22 1428.20 1429.21 1438.21 1444.20 1597.27 1638.28 1662.24 1668.22 1678.25 1686.24 1693.24
1398.20 1404.19 1412.20 1413.19
1413.19
1428.19
1428.20
1438.18 1444.15
1444.19
1597.24 1638.25 1662.19 1668.20
1597.24 1638.26
F. W. McLaffer& et al. /International Journal of Mass Spectrometry and Ion Processes 16.5 /166 Table 1 Mass 1701.26 1702.25 1708.24 1711.26 1716.26 1718.25 1727.26 1733.25 1742.26 1748.25 1751.27 1757.26 1758.25 1767.26 1775.28 1782.26 1783.26 1871.31 1910.32 1910.34 1920.27 1926.33 1927.32 1944.35 1951.34 1957.28 1967.29 1982.29 1990.30 1991.30 2000.31 2007.29 2014.31 2020.30 2022.29 2029.31 2030.31 2031.30 2037.29 2040.32 2055.32 2061.30 2070.32 2071.32 2072.30 2080.32 2087.31 2096.32 2199.38 2200.36 2214.30 2240.39 2286.33 2295.34
(1997) 457-466
461
(Continued) ‘3C”
Assignment A2T2C ATC2G T3CG A2C2G(I) A2T3(1)“51-55 TC2G2(11) AC2G2(1) T2CG201) ATCG2 T3G2(1)12-16 A2CG2 AT2G2 TCG301) ACGXI)” A3G2 ATG3 CG4(1) (10~6 (0~6 (II)a6 A3TCGZAH AT2C2G-P03H TC3G2-P03H A3C2G-P03H (Ua6 T3C3 TC4G(II) T2C3GfIIja A2T2C2(11)33-38 ATC3G A2C3GfI) TC3G2(11) A3T2C A2T4(11)11-16 T2C2G2(11) A3T3(1)50-55 A2T2CG(I)47-52 ATC2G2 T3CG2(1)12-17 A2C2G2(1) A3TCG2W AT3G2(1)11-16 A2T2G2(1)54-59 ATCG3 C264(1)29-34 A2CG3(1)27-32 TCG4(11)43-48 ACG4(1)28-33 (II)a7 (IIh7 A2T4G-AH Wa7 T3C3G(II)35-41 AT2C3G(IIja
IRMPDssI
IRMPDssII
IRMPDlds
1702.17 1711.37
1733.14
1727.26 1733.23 1742.26
1757.26 1758.26 1767.26
1783.22 1871.30 1910.31
1910.29 1920.20
IRMPD2ds 1701.26 1702.25 1708.24 1711.26 1716.24
1733.25 1742.26 1748.25 1751.27 1757.25 1758.27 1767.29 1775.29 1782.26 1783.26 1871.31 1910.30
BIRD ds
NS ds
1702.20
1702.24
1742.21
1711.00 1716.25 1718.24 1727.26 1733.22 1742.23
1757.26 1758.27
1910.20
1871.30 1910.31
1920.26 1926.31 1927.31 1944.32
1951.29
1951.35 1957.28 1982.14 1990.20 2000.31
1951.33 1957.28 1967.28 1990.30 1991.30 2000.32
1951.31 1957.30
2000.31
2007.21 2014.29 2020.28 2022.28
2014.32 2020.30 2022.30 2029.30 2030.31 2031.31
2020.26 2022.26 2030.36 2037.29
2037.28 2040.31 2055.34 2061.30 2070.33 2071.32
2071.26
2072.33
2072.29 2080.34 2087.34
2199.35 2200.31 2214.25 2286.23
2198.31
2199.34 2200.38
2240.36
2240.34 2286.33 2295.32
2087.34 2096.33 2198.28
2199.40
2241.28
2240.36
462 Table 1 Mass 2296.34 2319.36 2328.37 2341.34 2349.35 2360.36 2368.37 2384.36 2400.36 2512.44 2543.42 2569.44 2575.38 2583.40 2615.39 2632.41 2655.39 2673.41 2689.41 2695.40 2714.42 2802.49 2817.41 2865.43 2868.42 2883.50 2897.45 2929.44 2945.44 2946.47 3008.46 3027.48 3036.49 3042.47 3172.54 3177.52 3258.50 3292.51 3316.52 3339.54 3355.53 3427.56 3490.58 3498.50 3523.53 3547.54 3716.60 3723.64 3756.54 3787.57 3836.59 3870.15
F. W McLaffeq
et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466
(Continued) 13C”
0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Assignment TC4G2(11) A2T2C2G A3TC2G(II)48-54 T4CG2(11)15-21 A2T4G(II)ll-17 ATC2G3 A3TCG2(1) A2TCG3 ATCG4 (II)a8 (0~8 (I)a8 T3C4G(II) A2T3C3(11&12 T3C3G2 A3T2C2GtI) T3C2G3(11) A2TC2G3(1) ATC2G4(1)30-37 AT4G3(1)55-62 A2TCG4 (II)a9 A3T4CG-AH T3C5G A2TC2G4-AH (I)a9 A3T3C3(11) AT3C3G2 T3C3G3 A4T2C2G(I) A2T4G3(1) A3TCG4 (I)alO-AH (I)wlO-AH (1)alO (IxvlO AT3C3G3 (II)wll-AH A3TC3G4 (IXYll-GH (Ihvll-AH (I&11 (Ihvll T4C5G2(11) T3C5G3(11)52-62 AT3C4GXII) (IIhvl2 (II)al2 AT4C3G4-AH T4C6G2(11) AT3C5GXII) (IIhvl3-AH
-
IRMPDssI
IRMPDssII
IRMPDlds
IRMPD2ds
BIRD ds
2319.34 2328.36 2341.32 2349.35 2360.38 2368.36 2384.34 2400.36
2400.29
NS ds
2296.24
2349.26
2349.35
2360.35
2400.32 2512.41 2569.44
2569.45 2575.29 2583.27
2543.42 2569.43 2575.35 2583.39 2615.34 2632.40
2543.38 2569.39
2655.38 2673.41 2689.36 2695.39 2714.31 2802.40 2817.38 2865.37
2802.46
2689.40 2695.41 2714.41 2802.48
2868.34 2883.49
2802.42
2802.46
2883.39 2897.32 2945.44
2945.29 2946.45
2929.41 2945.40
2946.42
3172.49 3177.47
3042.45
3008.45 3027.43 3036.39 3042.46
3177.50
3177.51
3042.38 3172.45 3177.45
3042.43 3177.48
3258.35 3292.35 3316.49 3355.99
3355.43
3355.51
3490.54
3490.54
3339.49 3355.51
3427.41 3490.51 3523.44 3547.36 3716.42 3723.48 3756.38 3787.41
3723.50
3836.60 3870.46
3490.46 3498.50
3490.51
F. W. McLafferty et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466 Table 1
463
(Continued)
Mass
‘k,
Assignment
3891.59 3902.64 4005.65 4036.70 4076.62 4147.65 4149.65 4166.67 4276.68 4318.71 4361.76 4411.73 4497.71 4521.72 4574.74 4590.73 4632.76 4725.78 4823.79 4903.79 4945.82 5038.84 5112.80 5151.84 5287.90 5658.00 5810.94 5946.99 6253.03 6318.00 6438.05 6573.10 6586.02 6856.12 7056.15 7480.25 7674.25 7809.29 7987.31 8132.35 8525.36 9085.51 9128.47 9263.51 9399.57 11607.93
1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 5 6
AT4C3G4(11)37-48 (II)al3-AH (I&l3 (II)al3 T4C7G2(11)29-41 A4T5C3G(II)5-17 A2T3CSG3(11) A5T2C4G2(1)41-53 (Ihvl4-AH (IIhvl4 (II)wlS-2AH
IRMPDssI
4725.69
(I)w16 A2T4C5G5 (II)al7-AH (II)al7
5038.82
(IxY29 (Ihv30-2AH mv30-AH 0~30 mw37
IRMPD2ds
BIRD ds
NS ds
4005.60 4036.67
4276.68
4276.66
4166.69 4276.59 4318.56
4276.75
4411.67
4411.73
4411.59
4411.62
4632.52
4590.72 4632.73
4574.71 4590.71 4632.76 4725.80
4945.64
4903.76 4945.83
5151.56 5287.65
5112.75 5151.03 5287.85
4318.57 4361.43
0~15 (II)al5 (Ihvl6-AH (I&l6
(I)w24 (I)w25-AH (Ihv25 (I)w26-AH (I)a26 (II)w28-AH
4005.59
4276.55
4411.57
WV21 mw22-2AH mv22 (Ihv23-AH
IRMPDlds
3891.42 3902.46 4005.54 4036.51 4076.47 4147.49 4149.45
(Ihvl4 (II)wl5-AH (IIjwlS-CH &15-GH (0~15~AH mw15
0~18 m19-AH (0~19 (I)a20 A4T4C7G5 mw21-AH
IRMPDssII
4497.65 4521.51 4590.56
4902.65
5810.72 5946.74 6253.02
5810.88 5946.97
6438.00 6573.08
6438.07 6573.11
4590.70 4632.67 4725.64 4823.76
4574.74 4590.71 4632.87 4725.75
4903.83
5810.92
5151.65 5287.70 5658.78 5810.78 5946.75
5287.82 5658.91 5810.88 5946.97
6317.93 6573.00
6585.91 6855.90 7056.24 7480.11 7674.36 7809.45
7674.22 7809.24 7987.18 8132.29 8525.26 9085.34 9128.13 9263.15
9128.00
9128.17
9399.55 11607.92
a Another such sequence is not a possible product without the loss of T base.
would require T loss. The ions $T3G2), i(AT3G2) and j(A,T,G,) confirm (1)12-16, 11-16 and 54-59. The ion $A2T2CG) confirms the sequence (047-52; formation from the complementary
(11)13-18 would provide the same confirmation but require the loss of ‘*T. Similarly, $A,T,) confirms the complementary (050-55 and (II)lO-15, i(A,T,C,G,) confirms (041-53,
464
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Journal of Mass Spectrometry and Ion
Processes
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(1997)
457-466
B)
ds * base
860
1600 Fig. 2. (A) IRMPD
liO0 (50 ms) spectrum
m/z
loss
1400
of the ds 64-mer; (B) expansion
i(AT2C) confirms (11)9-12, i(A,T,) confirms (II)ll-16, i(T,CG,) confirms (11115-21, j(TCG,) confirms (11)43-48 and i(A,TC,G) confirms (1048-54. Identifying the precursor of i(A,T,C,G,) would be valuable in locating (dashed lines) the cleavages producing (1110-25 or (1034-49.
24-
DO of m/z
18’00
920-1050.
Of the internal ions found in the separate ss1 and ssI1 spectra, i(T,CG,) establishes (1112-17, j(ATC,G,) establishes (1)30-37, i(T2G2) establishes (11119-22, $T4C ,oZ) establishes (1029-41, i(A,T,C,) establishes (1033-38 and i(T,C,G) establishes (1035-41. Other j ions confirm the sequences (013-16, (1155-62, (11)5-12,
Fig. 3. 3, w and j ion assignments to the known sequence of the ds 64-mer (see [ll]). Dashed possibilities; heavy vertical lines of j ions are assignments from spectra of that ss1 precursor.
lines indicate
multiple
assignment
F. W. McLafseertyet al. /International Journal of Mass Spectrometry and Ion Processes 165/166
(1997) 457-466
465
r_____J
a ions
I
I
5’-‘l&GG&CCCGGG&_1__1.
I ‘30
r--___J
w
ions
CIXX>CGlllTA&4CGTCGlG%TGGGAAAA??-3,
3’-~GATCTCCT?GGGGCCCAT%9GCTCGAGCT%A~ti’&TGCij~~C~-5
w ions
L_____,
II
If
I
g
ions
A-Fig. 4. Ion assignments to the ds 70-mer sequence thought to be prepared by biological synthesis.
(105-17, (1037-48 and (1052-62. Cleavages identified only by i ions are indicated by vertical lines in Fig. 3 between the ss sequences. In summary, only the triplets 18-20(C2G) and 24-26(AG2) and the doublets 2L,22(AC) and 29,30(CG) of the ss1 sequence are not ordered. Further dissociation (MS/MS) of, for example, the i (I) 27-32 and j (II) 37-48 could complete the sequence.
4.2. ds 70-mer DNA from biological synthesis
A pair of ss 40-mer DNAs were synthesized with their last 10 3’-bases complementary to each other. After annealing, preparation of the fully complementary ds 70-mer DNA shown in Fig. 4 was attempted by 5’ to 3’ extension with Klenow fragment, a section of DNA polymerase I. Investigating its poor activity, the ESI/FTMS spectrum showed none of the expected molecular ions of the ds or ss components, indicating that full extension to the ds 70-mer had not been achieved (the ds molecular ions were the most abundant in the ds 64-mer spectrum) [19]. However, 5’-end a ions as large as (I)gX1 and (I&,, were observed (Fig. 41, consistent with a valid synthesis of the original 40-mer ss components. However, no 3’end w ions could be identified, again suggesting that the biological 5’ to 3’ extension was incomplete; the ‘ragged’ 3’-ends would give a multiplicity of w ions as well as molecular ions. The internal ion C,G, indicates that serious heterogeneity occurred only in the last 10 3’-end bases of ss-II, as C,G, can be assigned uniquely to (11154-60. The ion T,C,G, could arise from (1135-42 or (11145-52.
5. Conclusions For MS sequencing of DNA larger than 50- or 60-mers, double stranded DNA has already been shown to have the advantages of far more abundant molecular ions [17-19) and definitive base composition information [19]. The ds 64-mer data here also show that the base complementarity provides a valuable increase in sequence information from the combined fragmentation data of the two ss components. MS/MS can add detailed characterization of larger fragment ions [ll], giving promise that extensive MS sequence information can be obtained on much larger dsDNA, even at the sub-femtomole level [9,11]. Acknowledgements We thank Takeshi Sano for the Klenow fragment extension to prepare the 70-mer ds DNA and the National Institutes of Health (GM 16609) for generous financial support. References 111 M.J. Deery, S.G. Summerheld, A. Bury, K.R. Jennings. J. Am. Sot. Mass Spectrom. 8 (1997) 253-261. R. Venkataraghavan, P. Irving. Biochem. Biophys. Res. Commun. 39 (1970) 274. A.L. Cox, J. Shipper, Y. Chen, et al.. Science 264 (1994) 716-719. K. Biemann, I.A. Papayannopoulos. Act. Chem. Res. 27 (1994) 370-378. M.L. Gross. Act. Chem. Res. 27 (1994) 361-369. F.W. McLafferty. Act. Chem. Res. 27 (1994) 379-386. T.D. Wood, L.H. Chen, N.L. Kelleher, D.P. Little, G.L. Kenyon, F.W. McLafferty. Biochemistry 34 (1995) 16251-16254. N.L. Kelleher, C.A. Costello, T.P. Begley, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 6 (1995) 981-984.
121 F.W. McLafferty, 131 141 151
161 [71
@I
466 [9]
[lo] [ll] [12] [13] [14] [15] [16] [17]
F. W McLaffeerty et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466
G.A. Valaskovic, N.L. Kelleher, F.W. McLafferty. Science 273 (1996) 1199-1202. S.A. McLuckey, S.J. Habibi-Goudarzi. J. Am. Chem. Sot. 115 (1993) 12085-12095. D.P. Little, D.J. Aaserud, G.A. Valaskovic, F.W. McLafferty. J. Am. Chem. Sot. 118 (1996) 9352-9359. P.A. Limbach, P.F. Cram, J.A. McCloskey. J. Am Sot. Mass Spectrom. 6 (1995) 27-39. K.A. Sannes-Lowery, D.P. Mack, P. Hu, H.-Y. Mei, J.A. Loo. J. Am. Sot. Mass Spectrom. 8 (1997) 90-95. D.C. Muddiman, D.S. Wunschel, C. Liu, et al.. Anal. Chem. 68 (1996) 3705-3712. J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse. Science 246 (1989) 64-71. A.G. Marshall, P.B. Grosshans. Anal. Chem. 63 (1991) 215A-229A. Y. Naito, K. Ishikawa, Y. Koga, T. Tsuneyoshi, H. Terunuma, R. Arakawa. Rapid Commun. Mass Spectrom. 9 (1995) 1484-1486.
[18] D.S. Wunschel, K.F. Fox, A. Fox, J.E. Bruce, DC. Muddiman, R.D. Smith. Rapid Commun. Mass Spectrom. 10 (1996) 39-135. [19] D.J. Aaserud, N.L. Kelleher, D.P. Little, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 7 (1996) 1266-1269. [20] D.P. Little, J.P. Speir, M.W. Senko, P.B. O’Connor, F.W. McLafferty. Anal. Chem. 66 (1994) 2809-2815. [21] D.J. Aaserud, Z. Guan, D.P. Little, F.W. McLafferty, Int. J. Mass Spectrom. Ion Processes, in press. [22] W.D. Price, P.D. Schnier, E.R. Williams. Anal. Chem. 68 (1996) 859-866. [23] J.P. Speir, M.W. Senko, D.P. Little, J.A. Loo, F.W. McLafferty, J. Mass Spectrom. 30 (1995) 39-42. [24] D.P. Little, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 7 (1996) 209-210. [25] J.A. Loo, H.R. Udseth, R.D. Smith. Rapid Commun. Mass Spectrom. 2 (1988) 207-210.
International Journal of Mass Spectrometry and Ion Processes 165/166 (1997) 457-466
andIonProcesses
Double stranded DNA sequencing by tandem mass spectrometry Fred W. McLafferty”, David J. Aaserud, Ziqiang Guan, Daniel P. Little, Neil L. Kelleher Baker Chemistry Laboratory, Cornell Uniuersity, Ithaca, NY 14853-1301, USA
Received 28 April 1997; accepted 16 June 1997
Abstract Electrospray ionization produces far more abundant molecular ions for double stranded (ds) DNA than for single stranded (ss) and accurate molecular masses can provide the base composition of dsDNA. This study shows that the Fourier-transform mass spectra of a ds 64-mer DNA can provide approximately 50% more sequence information than the spectra of either individual ss component. Cleavages triggered by T loss are of low probability, but the opposite is true in the complementary ss strand that bears the base A at this site. The spectrum of the product of the attempted biological synthesis of a ds 70-mer DNA provided critical information on its extensive 3’-end heterogeneity. Published by Elsevier Science B.V. Keywords:
Electrospray;
Fourier-transform
MS; DNA; Tandem; Sequencing
1. Dedication It is a special privilege to contribute to an issue honoring Professor Keith Jennings, one of the true pioneers in molecular mass spectrometry.
*Corresponding author. Tel.: + 1 607 2554699;fax: + 1 607 2557880. 0168-3659/97/$17.00 Published by Elsevier Science B.V. PI1 SO168-1176(97)00171-7
For example, his prescient paper 3 decades ago demonstrated the collisionally activated dissociation (CAD) of benzene ions, while a very recent paper concerns CAD of the much larger biomolecule, bradykinin [ll. Hopefully, this paper on CAD of a 39-kDa double stranded DNA offers a useful extension of the unusual professional contributions of Keith Jennings to mass spectrometry.
458
F. W McLaffeq
et al. /International Journal of Mass Spectrometly and Ion Processes 165 / 166 (1997) 457-466
2. Introduction
ton dissociation (IRMPD) of the molecular ions. For the a, and w, ions of n = l-10, four were not found, of which three would have required T loss; for II = 11-20, 13 were not found, six with T loss; and for II = 21-30, 14 were not found, four with T loss. Because the most easily lost base, A, is the Watson-Crick complement of the least easily lost, T, backbone cleavages not represented in the ESI/FTMS spectrum of a single stranded (ss) DNA should have a relatively high probability of being represented in the spectrum of its ss complement. This is examined here with spectra of the ds DNA and its 64-mer ss complements used in the base composition study [19].
Sequencing of linear biomolecules by tandem mass spectrometry (MS/MS”) is well accepted. Its unique specificity and sensitivity are extensively documented for peptides [2-51 and larger proteins [6-91. Only recently, MS” sequencing has been extended to nucleotides (> lo-mers) [lo-141, made possible by the special advantages of gentle electrospray ionization (ES0 [15] and the high resolving power (> lo51 [6] of Fouriertransform @T) MS” 1161. ESI/FTMS has provided the complete sequence of a 50-mer DNA and extensive sequence data for DNA as large as a 108-mer [ill. Recent studies employing double-stranded (ds) DNA [14,17-191 show that ES1 yields abundant molecular ions of it and its two single strands (ss), whose exact masses can yield the base compositions of the ds and its ss components [19]. This derivation takes advantage of the Watson-Crick base complementarity of the two ss components (A and C in ss1 paired with T and G, respectively, in ssI1 and vice-versa). Here this complementary information is used to provide additional sequencing data for larger DNA molecules. The previous ESI/FTMS study of nine 42-108mer DNAs [ill showed that the most definitive sequence information comes from the terminal fragment ions. The 5, ions that contain the 5’terminus and the wn ions that contain the 3’terminus can be as large as 10 kDa to provide partial sequence information for up to approximately 30 bases from each end of the DNA molecule.’ The backbone cleavages forming the g, w and internal (i> ions are triggered by loss of an adjacent base, with base A loss favored [lO,ll]; however, base T loss is not found except in unusual cases (e.g. adjacent T bases) ill], so that the corresponding sequence fragment ion is not formed. This problem can be illustrated by a 50-mer DNA [ill for which the complete sequence could be derived from infrared multipho-
4.1. ds 64-mer
‘These a, ions were originally designated as a, +, - Base [9]; thus for gn, w,, and j, (internal ions), small II is the number of bases in the ion [IO].
ES1 ions from the annealed complementary 64-mers were trapped in the FTMS and subjected to IR laser [201 or BIRD 121,221heating to obtain molecular ions (M-1. These were abundant for
3. Experimental Procedures generally follow those reported recently [11,19-211. The complementary 64-mer DNAs and the 40-mer DNAs complementary in their first 10 bases (5’ vs. 3’) were synthesized and HPLC desalted at the Cornell Peptide/DNA Synthesis Facility and annealed for 5 min at 95°C. The ds 40-mers were then extended with Klenow fragment (a section of DNA polymerase I). Solutions were prepared as 5 PM (CH,CN-H,O, 3:l) with 0.02% piperidine and electrosprayed (- 2.5 kV, coaxial SF,) at 1 pl/min. The modified 6 T Finnigan FTMS instrument (Odyssey data system) consists of an external injection ES1 source with three rf-only quadrupoles for ion transportation into the trapped ion cell. Typically, ions were collected with the source/analyzer trap plates at -3 and -4 V and a N, pressure pulse (approx. 1O-6 torr) for enhanced trapping. For blackbody infrared radiative uissociation (BIRD) experiments [21,221, the traps were held at -2.5 V during the delay and reduced to - 1 V prior to broadband excitation and detection. 4. Results and discussion
F. W. McLaffeq
et al. /International Journal of Mass Spectromety and Ion Processes 165 / 166 (1997) 457-466
Fig. 1. Effect of IR laser irradiation time on ion yields from the ds 64-mer
both the double and single stranded (ds, ss> components whose mass values defined the base compositions of both ss DNA molecules [19]. The Mincrease with initial laser heating (Fig. 1) presumably resulted from ‘boil off of non-covalent adducts [21,23,24]. With increased IR irradiation, the ss M- ions abundances maximize before that of the ds M- ions, consistent with more strongly adducted ds M- ions and/or minimal fragmentation of ds M- ions to produce stable ss M- ions. IRMPD (50 ms, Fig. 2 and 60 ms> [20], BIRD [21] and nozzle-skimmer dissociation [25] spectra of ES1 ions from the ds sample and IRMPD spectra of ions from separate samples of ss1 and ssI1 gave the combined fragment mass data and sequence assignments of Table 1. None of the fragment masses of ds spectra was assignable to non-covalent complexes of fragments from both strands, although not all masses (omitted from Table 1) were structurally assignable. In such ds spectra, the fragments thus arise from either of the ss M- ions; the same interpretation problem was encountered for MS/MS of a mixture of 42 kDa proteins [S]. For the ss DNA mixture, molecular origin is clearly defined for fragment ions whose masses sum to that of one of the ss Mions; the peak masses 8132.29-4 &) plus 11607.92-6 (Wan)plus 135.05 (base AH lost in this fragmentation) sum to 19875.26-9 vs. the measured value 19874.33-9 for M- of SSI [19]. However, those fragment masses also formed from the individual ss DNAs (Table 1) clarify any ambiguity of their molecular parent. MS/MS spectra of
459
the individual ss1 and ssI1 molecular ions could provide similar delineations but were not measured here. Except for peaks of relatively small abundance, accuracies off 0.05 Da are found (Table 1) for most fragment masses up to approximately 2.5 kDa and k 0.2 Da up to 5 kDa without internal mass calibration. As found for ESI/FTMS of the 42 - to 108-mer DNAs [ll], this mass accuracy makes possible confident assignments of base composition (Fig. 3) for most fragments of up to five bases (however, note (Ixy6, A,T,CJ, = 1910.321 vs. (II&, A,T,C, = 1910.335). For higher masses, the accuracy of mass differences is higher, as is the assignment of composition differences. For ss1, a consistent set of 3’-end ions w4,
wg,
wX,
w1O,
wll>
w14,
w15,
w16,
w18,
w19:
w21,
are assignable and w30 (Table 11, with w3, (and _a,, its ss1 complement) assigned above (Fig. 3). Now the structural complementarity of ss1 and ssI1 (5’ to 3’, replacing A with T, C with G and vice versa) can provide further details using the ss-II 5’-end series; u2, a3, aI, a,, a12, al3 and al7 represent cleavages of complementary bonds not cleaved in ss1. The 5’-end ordering ‘C2T for both ss1 and ssI1 is indicated by the absence of an al peak consistent with the unfavorable loss of T [II]. Combining the complementary information for the 39-62 region of ss-I, only 42,43(CG)and 5R,59(AG)in ss-I are not ordered. Using this same approach for the first 16 bases of ss-I, the a ions assign sequences to the first 10 bases, while the w ions of ss-II assign the rest. For the 50-mer DNA [ll], internal j ions provided approximately 35% of the information on the sequence. Base compositions of j ions from spectra containing both ss components will be considered first to find any assignable to a unique position in only one of the ss strands. Thus i(A,CG,) fits only (1127-32, as formation of (11145-50 requires the loss of 44T. The ions i(ACG,) and i(C,G,) must represent (1128-33 and (1129-34, respectively. The ion i(AZCL) fits only (1143-46; both (1117-10 and (1050-53 require T loss. The ion i(A,TC,G) indicates (11)48-54; formation of (1145-51 and (1049-55
w23,
w24,
w25,
w26,
w29
460
F. W. McLaffeny et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466
Table 1 IRMPD (1, 50 ms; 2, 60 ms), BIRD and NS of ssI, ssI1 and ds-64 mer Mass 691.13 771.10 780.11 786.11 796.10 804.12 811.10 820.11 836.11 949.17 1004.19 1020.18 1060.15 1069.15 1084.15 1085.15 1090.14 1093.15 1099.15 1109.16 1115.15 1124.16 1125.15 1133.17 1140.15 1149.17 1238.15 1268.21 1349.23 1373.20 1379.19 1382.21 1388.20 1389.19 1398.21 1404.19 1412.21 1413.21 1419.19 1422.22 1428.21 1429.20 1438.21 1444.20 1453.21 1597.27 1638.28 1662.25 1668.23 1678.24 1686.26 1693.24
‘k”
Assignment
IRMPDssI
(I,II)a2 TC AC
691.11
(T-0 CG A2 TG AG G2
786.10
786.06
1004.15
Wa3 TC2(11)” AC2 ATC C2G T3 A2C AT2 ACG(I)” T2G ATG CG2 A2G TG2 AC2 ATC2-AH(H)
1020.12
(Ihv4 (I)a4 ATCXII) T3C A2C2(1)43-46 AT2C(II)9-12 TC2G01) AC2G(I)” T2CGtII) A2T2 ATCG T3G(1)13-16
1268.19 1349.20
Uhv5 Wa5 ATC3 T3C2 TC3GUI)” A2TC2(11) T2C2GtII)
691.10 771.06
804.10
(10~3 (II)a3
A2CG0) AT2G(II)14-17 TCG2 ACG2 T2G2(11)19-22 ATG2
IRMPDssII
IRMPDlds
771.09 780.10 786.09 796.09 804.10 811.09
949.16 1004.19 1020.18
1060.11
IRMPD2ds
BIRD ds
691.12 771.10 780.10 786.09 796.10 804.10 811.09 820.10 836.10 949.17
691.10
1020.18 1060.14 1069.15 1084.14 1085.15 1090.14 1093.15 1099.16
780.07 786.08 796.08
771.09 780.10 786.10 796.10
811.09
811.09
836.10 949.13 1004.18 1020.20
1099.13 1109.17
1109.16 1115.09 1124.15 1133.17
1268.21 1373.16 1379.16
1115.14 1124.15 1125.15 1133.16 1140.16 1149.15 1238.21 1268.24 1349.23 1373.20 1379.19
1115.13 1124.20 1133.17
1268.19
1398.19 1404.20
1413.15
1413.20
1428.14 1429.15
1429.19
1444.15
1444.00
1419.18
1453.20 1597.24 1638.25
1597.25 1638.28 1662.18 1668.23
1693.17
1268.20
1382.23
1382.25
1404.14
NS ds
1388.21 1389.21 1398.21 1404.20 1412.22 1413.21 1419.19 1422.22 1428.20 1429.21 1438.21 1444.20 1597.27 1638.28 1662.24 1668.22 1678.25 1686.24 1693.24
1398.20 1404.19 1412.20 1413.19
1413.19
1428.19
1428.20
1438.18 1444.15
1444.19
1597.24 1638.25 1662.19 1668.20
1597.24 1638.26
F. W. McLaffer& et al. /International Journal of Mass Spectrometry and Ion Processes 16.5 /166 Table 1 Mass 1701.26 1702.25 1708.24 1711.26 1716.26 1718.25 1727.26 1733.25 1742.26 1748.25 1751.27 1757.26 1758.25 1767.26 1775.28 1782.26 1783.26 1871.31 1910.32 1910.34 1920.27 1926.33 1927.32 1944.35 1951.34 1957.28 1967.29 1982.29 1990.30 1991.30 2000.31 2007.29 2014.31 2020.30 2022.29 2029.31 2030.31 2031.30 2037.29 2040.32 2055.32 2061.30 2070.32 2071.32 2072.30 2080.32 2087.31 2096.32 2199.38 2200.36 2214.30 2240.39 2286.33 2295.34
(1997) 457-466
461
(Continued) ‘3C”
Assignment A2T2C ATC2G T3CG A2C2G(I) A2T3(1)“51-55 TC2G2(11) AC2G2(1) T2CG201) ATCG2 T3G2(1)12-16 A2CG2 AT2G2 TCG301) ACGXI)” A3G2 ATG3 CG4(1) (10~6 (0~6 (II)a6 A3TCGZAH AT2C2G-P03H TC3G2-P03H A3C2G-P03H (Ua6 T3C3 TC4G(II) T2C3GfIIja A2T2C2(11)33-38 ATC3G A2C3GfI) TC3G2(11) A3T2C A2T4(11)11-16 T2C2G2(11) A3T3(1)50-55 A2T2CG(I)47-52 ATC2G2 T3CG2(1)12-17 A2C2G2(1) A3TCG2W AT3G2(1)11-16 A2T2G2(1)54-59 ATCG3 C264(1)29-34 A2CG3(1)27-32 TCG4(11)43-48 ACG4(1)28-33 (II)a7 (IIh7 A2T4G-AH Wa7 T3C3G(II)35-41 AT2C3G(IIja
IRMPDssI
IRMPDssII
IRMPDlds
1702.17 1711.37
1733.14
1727.26 1733.23 1742.26
1757.26 1758.26 1767.26
1783.22 1871.30 1910.31
1910.29 1920.20
IRMPD2ds 1701.26 1702.25 1708.24 1711.26 1716.24
1733.25 1742.26 1748.25 1751.27 1757.25 1758.27 1767.29 1775.29 1782.26 1783.26 1871.31 1910.30
BIRD ds
NS ds
1702.20
1702.24
1742.21
1711.00 1716.25 1718.24 1727.26 1733.22 1742.23
1757.26 1758.27
1910.20
1871.30 1910.31
1920.26 1926.31 1927.31 1944.32
1951.29
1951.35 1957.28 1982.14 1990.20 2000.31
1951.33 1957.28 1967.28 1990.30 1991.30 2000.32
1951.31 1957.30
2000.31
2007.21 2014.29 2020.28 2022.28
2014.32 2020.30 2022.30 2029.30 2030.31 2031.31
2020.26 2022.26 2030.36 2037.29
2037.28 2040.31 2055.34 2061.30 2070.33 2071.32
2071.26
2072.33
2072.29 2080.34 2087.34
2199.35 2200.31 2214.25 2286.23
2198.31
2199.34 2200.38
2240.36
2240.34 2286.33 2295.32
2087.34 2096.33 2198.28
2199.40
2241.28
2240.36
462 Table 1 Mass 2296.34 2319.36 2328.37 2341.34 2349.35 2360.36 2368.37 2384.36 2400.36 2512.44 2543.42 2569.44 2575.38 2583.40 2615.39 2632.41 2655.39 2673.41 2689.41 2695.40 2714.42 2802.49 2817.41 2865.43 2868.42 2883.50 2897.45 2929.44 2945.44 2946.47 3008.46 3027.48 3036.49 3042.47 3172.54 3177.52 3258.50 3292.51 3316.52 3339.54 3355.53 3427.56 3490.58 3498.50 3523.53 3547.54 3716.60 3723.64 3756.54 3787.57 3836.59 3870.15
F. W McLaffeq
et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466
(Continued) 13C”
0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Assignment TC4G2(11) A2T2C2G A3TC2G(II)48-54 T4CG2(11)15-21 A2T4G(II)ll-17 ATC2G3 A3TCG2(1) A2TCG3 ATCG4 (II)a8 (0~8 (I)a8 T3C4G(II) A2T3C3(11&12 T3C3G2 A3T2C2GtI) T3C2G3(11) A2TC2G3(1) ATC2G4(1)30-37 AT4G3(1)55-62 A2TCG4 (II)a9 A3T4CG-AH T3C5G A2TC2G4-AH (I)a9 A3T3C3(11) AT3C3G2 T3C3G3 A4T2C2G(I) A2T4G3(1) A3TCG4 (I)alO-AH (I)wlO-AH (1)alO (IxvlO AT3C3G3 (II)wll-AH A3TC3G4 (IXYll-GH (Ihvll-AH (I&11 (Ihvll T4C5G2(11) T3C5G3(11)52-62 AT3C4GXII) (IIhvl2 (II)al2 AT4C3G4-AH T4C6G2(11) AT3C5GXII) (IIhvl3-AH
-
IRMPDssI
IRMPDssII
IRMPDlds
IRMPD2ds
BIRD ds
2319.34 2328.36 2341.32 2349.35 2360.38 2368.36 2384.34 2400.36
2400.29
NS ds
2296.24
2349.26
2349.35
2360.35
2400.32 2512.41 2569.44
2569.45 2575.29 2583.27
2543.42 2569.43 2575.35 2583.39 2615.34 2632.40
2543.38 2569.39
2655.38 2673.41 2689.36 2695.39 2714.31 2802.40 2817.38 2865.37
2802.46
2689.40 2695.41 2714.41 2802.48
2868.34 2883.49
2802.42
2802.46
2883.39 2897.32 2945.44
2945.29 2946.45
2929.41 2945.40
2946.42
3172.49 3177.47
3042.45
3008.45 3027.43 3036.39 3042.46
3177.50
3177.51
3042.38 3172.45 3177.45
3042.43 3177.48
3258.35 3292.35 3316.49 3355.99
3355.43
3355.51
3490.54
3490.54
3339.49 3355.51
3427.41 3490.51 3523.44 3547.36 3716.42 3723.48 3756.38 3787.41
3723.50
3836.60 3870.46
3490.46 3498.50
3490.51
F. W. McLafferty et al. /International Journal of Mass Spectrometry and Ion Processes 165 / 166 (1997) 457-466 Table 1
463
(Continued)
Mass
‘k,
Assignment
3891.59 3902.64 4005.65 4036.70 4076.62 4147.65 4149.65 4166.67 4276.68 4318.71 4361.76 4411.73 4497.71 4521.72 4574.74 4590.73 4632.76 4725.78 4823.79 4903.79 4945.82 5038.84 5112.80 5151.84 5287.90 5658.00 5810.94 5946.99 6253.03 6318.00 6438.05 6573.10 6586.02 6856.12 7056.15 7480.25 7674.25 7809.29 7987.31 8132.35 8525.36 9085.51 9128.47 9263.51 9399.57 11607.93
1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 5 6
AT4C3G4(11)37-48 (II)al3-AH (I&l3 (II)al3 T4C7G2(11)29-41 A4T5C3G(II)5-17 A2T3CSG3(11) A5T2C4G2(1)41-53 (Ihvl4-AH (IIhvl4 (II)wlS-2AH
IRMPDssI
4725.69
(I)w16 A2T4C5G5 (II)al7-AH (II)al7
5038.82
(IxY29 (Ihv30-2AH mv30-AH 0~30 mw37
IRMPD2ds
BIRD ds
NS ds
4005.60 4036.67
4276.68
4276.66
4166.69 4276.59 4318.56
4276.75
4411.67
4411.73
4411.59
4411.62
4632.52
4590.72 4632.73
4574.71 4590.71 4632.76 4725.80
4945.64
4903.76 4945.83
5151.56 5287.65
5112.75 5151.03 5287.85
4318.57 4361.43
0~15 (II)al5 (Ihvl6-AH (I&l6
(I)w24 (I)w25-AH (Ihv25 (I)w26-AH (I)a26 (II)w28-AH
4005.59
4276.55
4411.57
WV21 mw22-2AH mv22 (Ihv23-AH
IRMPDlds
3891.42 3902.46 4005.54 4036.51 4076.47 4147.49 4149.45
(Ihvl4 (II)wl5-AH (IIjwlS-CH &15-GH (0~15~AH mw15
0~18 m19-AH (0~19 (I)a20 A4T4C7G5 mw21-AH
IRMPDssII
4497.65 4521.51 4590.56
4902.65
5810.72 5946.74 6253.02
5810.88 5946.97
6438.00 6573.08
6438.07 6573.11
4590.70 4632.67 4725.64 4823.76
4574.74 4590.71 4632.87 4725.75
4903.83
5810.92
5151.65 5287.70 5658.78 5810.78 5946.75
5287.82 5658.91 5810.88 5946.97
6317.93 6573.00
6585.91 6855.90 7056.24 7480.11 7674.36 7809.45
7674.22 7809.24 7987.18 8132.29 8525.26 9085.34 9128.13 9263.15
9128.00
9128.17
9399.55 11607.92
a Another such sequence is not a possible product without the loss of T base.
would require T loss. The ions $T3G2), i(AT3G2) and j(A,T,G,) confirm (1)12-16, 11-16 and 54-59. The ion $A2T2CG) confirms the sequence (047-52; formation from the complementary
(11)13-18 would provide the same confirmation but require the loss of ‘*T. Similarly, $A,T,) confirms the complementary (050-55 and (II)lO-15, i(A,T,C,G,) confirms (041-53,
464
F. W. McLaffegv et al. /International
Journal of Mass Spectrometry and Ion
Processes
I65 / 166
(1997)
457-466
B)
ds * base
860
1600 Fig. 2. (A) IRMPD
liO0 (50 ms) spectrum
m/z
loss
1400
of the ds 64-mer; (B) expansion
i(AT2C) confirms (11)9-12, i(A,T,) confirms (II)ll-16, i(T,CG,) confirms (11115-21, j(TCG,) confirms (11)43-48 and i(A,TC,G) confirms (1048-54. Identifying the precursor of i(A,T,C,G,) would be valuable in locating (dashed lines) the cleavages producing (1110-25 or (1034-49.
24-
DO of m/z
18’00
920-1050.
Of the internal ions found in the separate ss1 and ssI1 spectra, i(T,CG,) establishes (1112-17, j(ATC,G,) establishes (1)30-37, i(T2G2) establishes (11119-22, $T4C ,oZ) establishes (1029-41, i(A,T,C,) establishes (1033-38 and i(T,C,G) establishes (1035-41. Other j ions confirm the sequences (013-16, (1155-62, (11)5-12,
Fig. 3. 3, w and j ion assignments to the known sequence of the ds 64-mer (see [ll]). Dashed possibilities; heavy vertical lines of j ions are assignments from spectra of that ss1 precursor.
lines indicate
multiple
assignment
F. W. McLafseertyet al. /International Journal of Mass Spectrometry and Ion Processes 165/166
(1997) 457-466
465
r_____J
a ions
I
I
5’-‘l&GG&CCCGGG&_1__1.
I ‘30
r--___J
w
ions
CIXX>CGlllTA&4CGTCGlG%TGGGAAAA??-3,
3’-~GATCTCCT?GGGGCCCAT%9GCTCGAGCT%A~ti’&TGCij~~C~-5
w ions
L_____,
II
If
I
g
ions
A-Fig. 4. Ion assignments to the ds 70-mer sequence thought to be prepared by biological synthesis.
(105-17, (1037-48 and (1052-62. Cleavages identified only by i ions are indicated by vertical lines in Fig. 3 between the ss sequences. In summary, only the triplets 18-20(C2G) and 24-26(AG2) and the doublets 2L,22(AC) and 29,30(CG) of the ss1 sequence are not ordered. Further dissociation (MS/MS) of, for example, the i (I) 27-32 and j (II) 37-48 could complete the sequence.
4.2. ds 70-mer DNA from biological synthesis
A pair of ss 40-mer DNAs were synthesized with their last 10 3’-bases complementary to each other. After annealing, preparation of the fully complementary ds 70-mer DNA shown in Fig. 4 was attempted by 5’ to 3’ extension with Klenow fragment, a section of DNA polymerase I. Investigating its poor activity, the ESI/FTMS spectrum showed none of the expected molecular ions of the ds or ss components, indicating that full extension to the ds 70-mer had not been achieved (the ds molecular ions were the most abundant in the ds 64-mer spectrum) [19]. However, 5’-end a ions as large as (I)gX1 and (I&,, were observed (Fig. 41, consistent with a valid synthesis of the original 40-mer ss components. However, no 3’end w ions could be identified, again suggesting that the biological 5’ to 3’ extension was incomplete; the ‘ragged’ 3’-ends would give a multiplicity of w ions as well as molecular ions. The internal ion C,G, indicates that serious heterogeneity occurred only in the last 10 3’-end bases of ss-II, as C,G, can be assigned uniquely to (11154-60. The ion T,C,G, could arise from (1135-42 or (11145-52.
5. Conclusions For MS sequencing of DNA larger than 50- or 60-mers, double stranded DNA has already been shown to have the advantages of far more abundant molecular ions [17-19) and definitive base composition information [19]. The ds 64-mer data here also show that the base complementarity provides a valuable increase in sequence information from the combined fragmentation data of the two ss components. MS/MS can add detailed characterization of larger fragment ions [ll], giving promise that extensive MS sequence information can be obtained on much larger dsDNA, even at the sub-femtomole level [9,11]. Acknowledgements We thank Takeshi Sano for the Klenow fragment extension to prepare the 70-mer ds DNA and the National Institutes of Health (GM 16609) for generous financial support. References 111 M.J. Deery, S.G. Summerheld, A. Bury, K.R. Jennings. J. Am. Sot. Mass Spectrom. 8 (1997) 253-261. R. Venkataraghavan, P. Irving. Biochem. Biophys. Res. Commun. 39 (1970) 274. A.L. Cox, J. Shipper, Y. Chen, et al.. Science 264 (1994) 716-719. K. Biemann, I.A. Papayannopoulos. Act. Chem. Res. 27 (1994) 370-378. M.L. Gross. Act. Chem. Res. 27 (1994) 361-369. F.W. McLafferty. Act. Chem. Res. 27 (1994) 379-386. T.D. Wood, L.H. Chen, N.L. Kelleher, D.P. Little, G.L. Kenyon, F.W. McLafferty. Biochemistry 34 (1995) 16251-16254. N.L. Kelleher, C.A. Costello, T.P. Begley, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 6 (1995) 981-984.
121 F.W. McLafferty, 131 141 151
161 [71
@I
466 [9]
[lo] [ll] [12] [13] [14] [15] [16] [17]
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[18] D.S. Wunschel, K.F. Fox, A. Fox, J.E. Bruce, DC. Muddiman, R.D. Smith. Rapid Commun. Mass Spectrom. 10 (1996) 39-135. [19] D.J. Aaserud, N.L. Kelleher, D.P. Little, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 7 (1996) 1266-1269. [20] D.P. Little, J.P. Speir, M.W. Senko, P.B. O’Connor, F.W. McLafferty. Anal. Chem. 66 (1994) 2809-2815. [21] D.J. Aaserud, Z. Guan, D.P. Little, F.W. McLafferty, Int. J. Mass Spectrom. Ion Processes, in press. [22] W.D. Price, P.D. Schnier, E.R. Williams. Anal. Chem. 68 (1996) 859-866. [23] J.P. Speir, M.W. Senko, D.P. Little, J.A. Loo, F.W. McLafferty, J. Mass Spectrom. 30 (1995) 39-42. [24] D.P. Little, F.W. McLafferty. J. Am. Sot. Mass Spectrom. 7 (1996) 209-210. [25] J.A. Loo, H.R. Udseth, R.D. Smith. Rapid Commun. Mass Spectrom. 2 (1988) 207-210.