Q-TOF MS/MS : de-novo sequencing

Tip 1. Generally, Q-TOF ESI-MS shows multiple charged ions in the mass spectrum e.g. 729(+2) = m/z



m/z 729.8 = [M+2H]/2



M=(729.8x2)-2=1457.6

2. MS/MS fragmented ions : next page 3.

Leu(L)=Ile(I)

and Phe(F)=Met(M) oxidation have the same MW ; It means that L or F can be replaced with I or M, respectively.

IN2_MD_44_729 MaxEnt 3 5 [ Ev - 141588,It 50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 V L R A D V M E F M T D M DTFMDR E V A 1246.47

y 10 L 1: TOF MSMS 729.80ES+ V bMax y Max 1076.37

y 8 185.14

a2 213.14

b2 284.17

b3 947.34

y 7 % 175.10

y 1 0 143.10

86.08

L 100 200 1175.41

y 9 685.27

y 5 800.28

y 6 300 355.24

a4 437.17

y 3 512.27

b5 584.23

y 4 667.25

730.27

400 500 600 700 801.36 929.36

800 900 1247.35

1058.34

1040.33

1000 1077.47

1078.23

1100 1200 1458.67(M+H) + 1459.85

1248.611359.52

y 11 1256.47

1300 1400 1460.60

M/ z 1500

y ions

Fragmentation of Peptides

2D 17=18=44~48 (spot No.) ; 710(+3) ([M+3H] 3+ ) - NLLLQGDAHLDPNVLQLTR (estimated sequen.)

IN2_MD_44_710 MaxEnt 3 8 [Ev - 264821,It50,En1] (0.050,200.00,0.200,1400.00,3,Cmp) 100 NL R T 200.12

a2 L L L Q L V QGDAHLD N P 940.46

y 8 D L H PN A D G Q V LQLTR L L 1: TOF MSMS 710.66ES+ L N bMax y Max 228.11

b2 341.19

b3 1789.69

y 16 1902.84

y 17 % 1055.47

y 9 0 183.09

155.10

282.15

492.14

517.26

y 4 630.35

y 5 735.30

720.22

200 400 600 895.44

800 1168.55

y 10 1305.58

y 11 1548.63

y 14 1659.62

951.83

1056.58

1169.66

1376.64

y 12 1491.68

y 13 1000 1200 1400 1600 1676.73

y 15 1800 1904.03

2025.85

2000 2109.40

2134.04

M/ z

2D 17=18=44~48 ; 729(+2) - VLAVEMDTFMDR

IN2_MD_44_729 MaxEnt 3 5 [Ev - 141588,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) V L R A D V M E F M T D 100 M DTFMDR E V A 1246.47

y 10 L 1: TOF MSMS 729.80ES+ V bMax y Max 1076.37

y 8 185.14

a2 213.14

b2 284.17

b3 947.34

y 7 % 175.10

y 1 0 143.10

86.08

L 100 200 1175.41

y 9 685.27

y 5 800.28

y 6 300 355.24

a4 437.17

y 3 512.27

b5 584.23

y 4 667.25

730.27

400 500 600 700 801.36 929.36

800 900 1247.35

1058.34

1040.33

1000 1077.47

1078.23

1100 1200 1458.67(M+H) + 1248.611359.52

y 11 1256.47

1300 1400 1459.85

1460.60

M/ z 1500

2D 17=18=44~48 ; 895(+2) - LQGDAHLDPNDPQLTR

IN2_MD_44_895 MaxEnt 3 10 [Ev - 37555,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) R LQ T GD L A Q H PD L D N P PN D 100 L DP H Q A L D G 1: TOF MSMS 895.41ES+ TR Q L bMax y Max 1789.78(M+H) + 940.47

y 8 % 0 110.06

H 209.09

200 1055.47

y 9 381.13

400 594.24

a6 517.26;y 4 622.23

b6 600 735.30

b7 850.32

b8 895.29

743.22

800 941.57

942.35

1000 1168.52

y 10 1056.58

1160.42

1169.63

1243.62

1200 1305.62

y 11 1790.59

1548.67

y 14 1376.64

y 12 1400 1491.70

y 13 1549.51

1596.70

1600 1789.03

1771.81

1792.22

1799.93

M/ z 1800

2D 16=26=27 ?

IN2_MD_51 9 (0.782) Sm (SG, 2x5.00); Cm (4:22) 954.65

100 955.66

968.68

% 861.55

874.43

902.60

940.64

938.63

970.68

984.68

986.67 1006.91

2D 26=27

1042.43

1043.41

1089.08

1102.51

1108.70

0 IN2_MD_43 9 (0.781) Sm (SG, 2x5.00); Cm (3:9) 100 954.66

968.68

970.68

2D 16

% 858.36

875.43

904.62

916.57

942.42

984.68

985.68

986.691002.62

TOF MS ES+ 1.16e3

1158.78

TOF MS ES+ 210 1042.43

1089.41

1042.93

1062.60

1102.52

1142.47

1166.77

0 860 880 900 920 940 960 980 1000 1020 1040 1060 1080 1100 1120 1140 1160 m/z

2D 30 ; 794(+2) - VGSYQGEELYAPFK

IN2_MD_54_794 MaxEnt 3 5 [Ev 17687,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) V G K S F Y P A Q G Y E L E EL 100 E Y G Q A P Y 1053.60

y 9 F S 1: TOF MSMS 794.44ES+ G K bMax V y Max 1587.87(M+H) + 794.53

1588.70

877.48

% 391.27

y 3 795.41

1181.64

y 10 787.50

0 133.10

244.15

b3 136.08

Y 226.14

379.22

292.15

a4 361.21

86.09

L 100 200 300 400 462.31

y 4 530.36

500 625.39

y 5 607.30

600 686.47

756.39

700 867.53

y 7 877.98

927.04

983.56

1164.60

1054.50

800 900 1000 1057.62

1100 1590.19

1182.54

1569.73

1249.66

1327.79

1371.66

1475.81

1510.70

1200 1300 1400 1500 1593.61

1597.59

1600.79

M/ z 1600

2D 33 ; 1012(+2) - DLSQDDGLVPLVEEL~~~

IN2_MD_57_1012 MaxEnt 3 6 [Ev - 9400,It50,En1] (0.050,200.00,0.200,1400.00,3,Cmp) 100 (1202.50) DLSQDDGLV PLV EEL(1202.50) L EE V L P 1901.93

y 6 V L GD D Q S L 1: TOF MSMS 1012.25ES+ D (190.63) bMax y Max 1463.75

% 306.15

1903.08

783.39

235.12

391.24 439.27

0 211.14

207.12

200 400 732.96

600 800 951.57

1011.50

1133.56

1237.67

1000 1200 1400 2616.16

y 13 1592.75

y 3 1600 1691.83

y 4 1900.80

1883.97

1800 2001.09

2000 2171.22

2200 2401.12

y 11 2615.41

2400 2617.37

2600 2851.93

2853.22

2800 2926.11

3036.96

3049.87

M/ z 3000

2D 36=38 ; 1194(+2) - ~~~NGDLTTLADVVELK

IN2_MD_59_1194 MaxEnt 3 19 [Ev 87850,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 Q L E (899.38) V V D A L T NG T DL L D 1194.45

G TTL N AD V V (899.38) EL 1: TOF MSMS 0.00ES+ Q bMax y Max % 215.12

284.14

436.21

553.26

587.29

y 5 129.08

773.36

y 7 1817.62

1818.48

2386.69

987.51

y 9 943.39

907.35

1088.51

y 10 1201.59

y 11 1373.65

y 13 1487.61

y 14 1716.68

1545.58

1917.66

1973.17

1987.61

2173.80

2006.69

2175.19

2175.86

2351.00

2388.28

2398.56

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 M/ z 2400

2D 39~43 ; 1102(+3) - ~~~TSDLGDPNYPHLGLDYF~~~

IN2_MD_61_1102 MaxEnt 3 7 [Ev - 13525,It50,En1] (0.050,200.00,0.200,1400.00,3,Cmp) 100 (259.08) F (1121.47) Y D L L H T S D P Y L N P 1594.55

y 11 D D L PNYPHL D S T GLDY (1121.47) 1: TOF MSMS 1102.10ES+ F(259.08) bMax y Max 284.16

% 213.13

1220.51

y 8 0 185.15

250 873.36

y 5 355.22

588.25

y 2 447.15

797.85

703.29

y 3 1141.88

1221.41

1396.80

500 750 1000 1250 1500 1766.54

y 13 1879.68

y 14 2282.54

2081.75

y 16 2282.80

2921.81

2792.80

2645.71

2530.75

2429.69

3091.64

3187.57

1750 2000 2250 2500 2750 3000 3306.09

3306.56

3315.61

M/ z 3250

2D 54 ; 842(+2) - NTVDDGSPLLLSSPLR

IN2_MD_66_842 MaxEnt 3 7 [Ev - 33530,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 R L NTV DD P S S G L S L PL L L P 995.70

y 9 S L G D D SSPLR V 1: TOF MSMS 842.54ES+ T N bMax y Max % 0 217.10

189.10

157.08

200 316.18

219.19

400 559.37

y 5 672.46

y 6 385.29

y 3 472.36

y 4 431.20 555.20

573.22

600 690.30

785.55

726.48

y 7 842.63

800 843.49

898.61;y 8 1082.77

y 10 996.58

1369.85

1139.78

y 11 1254.82

y 12 1253.73

1236.84

1368.74

a14 1351.73

y 13 1468.89

y 14 1469.76

1000 1200 1400 1683.86(M+H) + 1667.05

1684.63

1691.00

M/ z 1600

2D 54 ; 870(+2) - LLVATEDNPFVVMFK

IN2_MD_66_870 MaxEnt 3 14 [Ev - 40632,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 K LL 227.19

b2 F V A M T V E V D F N P P N F D V E V T A V MFK 1: TOF MSMS 870.03ES+ LL bMax y Max % 0 199.20

a2 147.12

y 1 200 883.56

y 7 326.27

b3 441.25

y 3 540.33

y 4 397.32

b4 400 1342.66

639.40

y 5 739.42

786.41

y 6 819.55

1112.58

y 9 997.62

y 8 884.48

933.61

1048.65

1241.65

1177.66

y 10 600 800 1000 1200 y 11 1413.73

y 12 1414.87

1400 1512.84

y 13 1513.71

1572.04

1600 1739.99

1764.87

1798.75

M/ z 1800

2D 60=61 ; 627(+2) - MLGLFNDASDR

IN2_MD_72_627 MaxEnt 3 7 [Ev - 16880,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 R ML D G S L A D FND N 628.35

F L G ASDR L 1: TOF MSMS 627.73ES+ M bMax y Max 219.17

994.38

y 9 % 233.14

a2 824.26

y 7 0 120.07

a1 F 86.07

100 175.10

y 1 200 261.13

b2 318.14

b3 300 410.24

448.17

y 4 403.23

a4 467.30

627.41

563.18

y 5 400 500 600 995.29

629.25

678.15

785.52

825.35

937.31

a9 y 8 895.45

700 800 900 996.49

1000 1107.42

y 10 1100 1255.52

1253.61

1257.91

1262.57

1137.65

1200 M/ z 1300

2D 71 ; 960(+2) - WAMoxLGALGCVF~~

IN2_MD_79_960 MaxEnt 3 10 [Ev - 34656,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) WAF L G A P L G F C V V C F G 100 365.14

L P A G 1161.71

y 5 L F 1: TOF MSMS 960.59ES+ AW bMax y Max 381.33

% 0 1274.78

441.17

453.21

623.98

646.34

b6 698.50

y 1 761.21

845.55

y 2 944.62

y 3 960.58

991.69

1104.67

y 4 1162.60

y 6 1402.75

y 8 1919.90(M+H) + 1345.88

y 7 1403.91

1408.88

1515.84

y 9 1631.07 1689.96

1904.15

1881.56

1921.10

1955.22

1977.80

1980.27

M/ z 400 600 800 1000 1200 1400 1600 1800

2D 74 ; 704(+2) - LTYYTPEYETK

IN2_MD_82_704 MaxEnt 3 5 [Ev - 44667,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 100 K LT T Y E Y Y T E P P 766.44

y 6 E T Y Y Y ETK T 1: TOF MSMS 704.41ES+ L bMax y Max 867.50

y 7 1030.56

y 8 1193.59

y 9 704.54

% 0 86.10

a1 L 100 136.08

Y 219.23

299.16

360.23

237.14

428.23

540.31

y 4 519.25

620.31

200 300 400 500 600 700 767.36

868.41

b7 1012.55

849.51

946.51

971.80

1031.46

b8 1032.66

800 900 1000 1100 1194.70

1407.82(M+H) + 1408.30

1409.69

1294.70

y 10 1276.65

1389.71

1200 1300 1410.39

1411.88

M/ z 1400

2D 77 ; 998(+2) – VMPGLLNSSPN~~(?)

IN2_MD_85_998 MaxEnt 3 29 [Ev 35938,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) V M PG L (866.32) L N S S P N N P 100 S S N L (866.32) L G P 1765.01

y 9 1: TOF MSMS 998.06ES+ M V bMax y Max % 1765.87

0 219.20

365.14

268.19

353.30

480.31

643.40

708.43

761.50

882.89

998.67

1096.60

y 2 883.59

884.15

997.85

980.66

1183.64

y 3 155.10

200 400 600 800 1000 1200 1996.08

1384.71

y 5 1994.90(M+H) + 1270.56

y 4 1400 1497.71

y 6 1633.27

1600 1701.02

1994.50

1767.05

1774.60

1800 1997.51

1998.52

M/ z 2000

2D 79 ; 727(+2) – WLSMQVARVVHK

IN2_MD_86_727 MaxEnt 3 18 [Ev - 113933,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) W KH L S V V R A V 100 MQV ARV V HK Q 954.62

M S LW 1: TOF MSMS 727.50ES+ bMax y Max 727.56

% 0 369.25

173.15

201.14

219.23

300.22

b2 365.26

383.27

y 3 482.34

y 4 464.33

509.36 638.36

y 5 709.43

y 6 157.11

728.47

742.59

808.51

y 7 953.67

873.70

955.52

956.74

1067.71

y 9 1154.76

y 10 1253.851275.83

1453.80(M+H) + 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 M/ z 1500

2D 21=33=79 ; have same peptide m/z 727 (MW=1454) - aldolase

IN2_MD_57_727 4 (0.358) Sm (SG, 2x5.00); Cm (3:10) 100 954.58

955.58

% 201.13

369.22

173.14

202.14

365.24

482.32

507.27

638.40

0 IN2_MD_48_727 3 (0.271) Sm (SG, 2x5.00); Cm (3:12) 727.50

742.48

727.40

100 % 173.11

201.10

220.15

369.18

365.20

383.19

464.26

638.23

0 IN2_MD_86_727 18 (1.563) Sm (SG, 2x5.00); Cm (4:35) 709.32

728.43

742.40

953.61

956.60

808.38

954.46

955.45

956.50

100 954.61

955.62

728.59

% 173.14

201.14

369.25

365.25

383.27 482.34

709.42

729.44

808.50

953.63

956.64

0 100 200 300 400 500 600 700 800 900 1000 1154.71

1155.54

1154.72

1100 1200 TOF MSMS 727.46ES+ 49.0

2D 33

TOF MSMS 727.38ES+ 24.8

2D 21

TOF MSMS 727.50ES+ 187

2D 79

1300 1400 m/z