PEAKPLOT: Visualizing Fragmented Peptide Mass Spectra in Proteomics - - PowerPoint PPT Presentation

SLIDE 1

PEAKPLOT: Visualizing Fragmented Peptide Mass Spectra in Proteomics

Christian Panse

Bertran Gerrits Ralph Schlapbach July 8, useR-2009, Agrocampus-Ouest, Rennes, France

SLIDE 2

goals of shotgun proteomics

◮ identification ◮ quantification ◮ detection and discovery of post translational modifications

SLIDE 3

method of choice: mass spectrometry

ion source

SLIDE 4

method of choice: mass spectrometry

ion source mass analyzer

SLIDE 5

method of choice: mass spectrometry

ion source mass analyzer detector source: FGCZ, http://en.wikipedia.org/wiki/Mass spectrometry

SLIDE 6

slide by Jonas Grossmann jg@fgcz.ethz.ch

SLIDE 7

peptide fragmentation into b and y ions

SLIDE 8

why peakplot?

◮ annotated spectra important for quality control

SLIDE 9

why peakplot?

◮ annotated spectra important for quality control ◮ annotated spectra required for publication and reviewing

purposes

SLIDE 10

why peakplot?

◮ annotated spectra important for quality control ◮ annotated spectra required for publication and reviewing

purposes

◮ no manual annotation/validation of individual spectra is

feasible

SLIDE 11

why peakplot?

◮ annotated spectra important for quality control ◮ annotated spectra required for publication and reviewing

purposes

◮ no manual annotation/validation of individual spectra is

feasible

◮ existing software has limitations

not open, not scalable, too complex to install

SLIDE 12

peakplot input

file containing:

◮ mass spectrum ◮ peptide sequence

SLIDE 13

peakplot input

file containing:

◮ mass spectrum ◮ peptide sequence

SLIDE 14

step 0: parsing the input data

reqired attributes:

◮ peptide query# and peptide hit# ◮ MS/MS ◮ assigned peptide sequence ◮ score or e-value ◮ peptide modification

SLIDE 15

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

SLIDE 16

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590

SLIDE 17

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590 letter a a* a 0 b b* b 0 y y* y 0 z+1 z+2 b-98 b*-98 b 0-98 y-98 y*-98 y 0-98 letter L[1] 86.10 114.09 2291.08 2274.06 2273.07 2276.07

• 0.91
• 1.90

2193.11 2176.08 2175.10 L[18] I[2] 199.18 227.18 2178.00 2160.97 2159.99 2162.99 112.17 111.19 2080.02 2063.00 2062.01 I[17] Q[3] 327.24 310.21 355.23 338.21 2064.92 2047.89 2046.91 2049.91 2050.91 240.23 239.25 1966.94 1949.91 1948.93 Q[16] Q[4] 455.30 438.27 483.29 466.27 1936.86 1919.83 1918.85 1921.85 1922.85 368.29 367.31 1838.88 1821.85 1820.87 Q[15] L[5] 568.38 551.36 596.38 579.35 1808.80 1791.77 1790.79 1793.79 1794.80 481.37 480.39 1710.82 1693.80 1692.81 L[14] S[6] 735.38 718.35 717.37 763.37 746.35 745.36 1695.71 1678.69 1677.70 1680.70 1681.71 665.40 648.37 647.39 1597.74 1580.71 1579.73 S[13] K[7] 863.48 846.45 845.46 891.47 874.44 873.46 1528.72 1511.69 1510.71 1513.71 1514.71 793.49 776.47 775.48 1430.74 1413.71 1412.73 K[12] D[8] 978.50 961.48 960.49 1006.50 989.47 988.49 1400.62 1383.60 1382.61 1385.61 1386.62 908.52 891.49 890.51 1302.64 1285.62 1284.63 D[11] D[9] 1093.53 1076.50 1075.52 1121.52 1104.50 1103.51 1285.59 1268.57 1267.58 1270.58 1271.59 1023.55 1006.52 1005.54 1187.62 1170.59 1169.61 D[10] F[10] 1240.60 1223.57 1222.59 1268.59 1251.57 1250.58 1170.57 1153.54 1152.56 1155.56 1156.56 1170.62 1153.59 1152.60 1072.59 1055.56 1054.58 F[9] D[11] 1355.62 1338.60 1337.61 1383.62 1366.59 1365.61 1023.50 1006.47 1005.49 1008.49 1009.50 1285.64 1268.62 1267.63 925.52 908.50 907.51 D[8] E[12] 1484.67 1467.64 1466.66 1512.66 1495.64 1494.65 908.47 891.45 890.46 893.46 894.47 1414.68 1397.66 1396.67 810.50 793.47 792.48 E[7] D[13] 1599.69 1582.67 1581.68 1627.69 1610.66 1609.68 779.43 762.40 761.42 764.42 765.43 1529.71 1512.69 1511.70 681.45 664.43 663.44 D[6] Y[14] 1762.76 1745.73 1744.75 1790.75 1773.73 1772.74 664.40 647.38 649.39 650.40 1692.78 1675.75 1674.76 549.40 548.42 Y[5] L[15] 1875.84 1858.81 1857.83 1903.84 1886.81 1885.83 501.34 484.31 486.33 487.34 1805.86 1788.83 1787.85 386.34 385.35 L[4] L[16] 1988.93 1971.90 1970.91 2016.92 1999.89 1998.91 388.26 371.23 373.24 374.25 1918.94 1901.92 1900.93 273.25 272.27 L[3] Q[17] 2116.98 2099.96 2098.97 2144.98 2127.95 2126.97 275.17 258.14 260.16 261.17 2047.00 2029.98 2028.99 160.17 159.18 Q[2] K[18] 2245.08 2228.05 2227.07 2273.07 2256.05 2255.06 147.11 130.09 132.10 133.11 2175.10 2158.07 2157.09 32.11 31.13 K[1]

SLIDE 18

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590 letter a a* a 0 b b* b 0 y y* y 0 z+1 z+2 b-98 b*-98 b 0-98 y-98 y*-98 y 0-98 letter L[1] 86.10 114.09 2291.08 2274.06 2273.07 2276.07

• 0.91
• 1.90

2193.11 2176.08 2175.10 L[18] I[2] 199.18 227.18 2178.00 2160.97 2159.99 2162.99 112.17 111.19 2080.02 2063.00 2062.01 I[17] Q[3] 327.24 310.21 355.23 338.21 2064.92 2047.89 2046.91 2049.91 2050.91 240.23 239.25 1966.94 1949.91 1948.93 Q[16] Q[4] 455.30 438.27 483.29 466.27 1936.86 1919.83 1918.85 1921.85 1922.85 368.29 367.31 1838.88 1821.85 1820.87 Q[15] L[5] 568.38 551.36 596.38 579.35 1808.80 1791.77 1790.79 1793.79 1794.80 481.37 480.39 1710.82 1693.80 1692.81 L[14] S[6] 735.38 718.35 717.37 763.37 746.35 745.36 1695.71 1678.69 1677.70 1680.70 1681.71 665.40 648.37 647.39 1597.74 1580.71 1579.73 S[13] K[7] 863.48 846.45 845.46 891.47 874.44 873.46 1528.72 1511.69 1510.71 1513.71 1514.71 793.49 776.47 775.48 1430.74 1413.71 1412.73 K[12] D[8] 978.50 961.48 960.49 1006.50 989.47 988.49 1400.62 1383.60 1382.61 1385.61 1386.62 908.52 891.49 890.51 1302.64 1285.62 1284.63 D[11] D[9] 1093.53 1076.50 1075.52 1121.52 1104.50 1103.51 1285.59 1268.57 1267.58 1270.58 1271.59 1023.55 1006.52 1005.54 1187.62 1170.59 1169.61 D[10] F[10] 1240.60 1223.57 1222.59 1268.59 1251.57 1250.58 1170.57 1153.54 1152.56 1155.56 1156.56 1170.62 1153.59 1152.60 1072.59 1055.56 1054.58 F[9] D[11] 1355.62 1338.60 1337.61 1383.62 1366.59 1365.61 1023.50 1006.47 1005.49 1008.49 1009.50 1285.64 1268.62 1267.63 925.52 908.50 907.51 D[8] E[12] 1484.67 1467.64 1466.66 1512.66 1495.64 1494.65 908.47 891.45 890.46 893.46 894.47 1414.68 1397.66 1396.67 810.50 793.47 792.48 E[7] D[13] 1599.69 1582.67 1581.68 1627.69 1610.66 1609.68 779.43 762.40 761.42 764.42 765.43 1529.71 1512.69 1511.70 681.45 664.43 663.44 D[6] Y[14] 1762.76 1745.73 1744.75 1790.75 1773.73 1772.74 664.40 647.38 649.39 650.40 1692.78 1675.75 1674.76 549.40 548.42 Y[5] L[15] 1875.84 1858.81 1857.83 1903.84 1886.81 1885.83 501.34 484.31 486.33 487.34 1805.86 1788.83 1787.85 386.34 385.35 L[4] L[16] 1988.93 1971.90 1970.91 2016.92 1999.89 1998.91 388.26 371.23 373.24 374.25 1918.94 1901.92 1900.93 273.25 272.27 L[3] Q[17] 2116.98 2099.96 2098.97 2144.98 2127.95 2126.97 275.17 258.14 260.16 261.17 2047.00 2029.98 2028.99 160.17 159.18 Q[2] K[18] 2245.08 2228.05 2227.07 2273.07 2256.05 2255.06 147.11 130.09 132.10 133.11 2175.10 2158.07 2157.09 32.11 31.13 K[1]

m/z 500 1000 1500 2000

SLIDE 19

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590 letter a a* a 0 b b* b 0 y y* y 0 z+1 z+2 b-98 b*-98 b 0-98 y-98 y*-98 y 0-98 letter L[1] 86.10 114.09 2291.08 2274.06 2273.07 2276.07

• 0.91
• 1.90

2193.11 2176.08 2175.10 L[18] I[2] 199.18 227.18 2178.00 2160.97 2159.99 2162.99 112.17 111.19 2080.02 2063.00 2062.01 I[17] Q[3] 327.24 310.21 355.23 338.21 2064.92 2047.89 2046.91 2049.91 2050.91 240.23 239.25 1966.94 1949.91 1948.93 Q[16] Q[4] 455.30 438.27 483.29 466.27 1936.86 1919.83 1918.85 1921.85 1922.85 368.29 367.31 1838.88 1821.85 1820.87 Q[15] L[5] 568.38 551.36 596.38 579.35 1808.80 1791.77 1790.79 1793.79 1794.80 481.37 480.39 1710.82 1693.80 1692.81 L[14] S[6] 735.38 718.35 717.37 763.37 746.35 745.36 1695.71 1678.69 1677.70 1680.70 1681.71 665.40 648.37 647.39 1597.74 1580.71 1579.73 S[13] K[7] 863.48 846.45 845.46 891.47 874.44 873.46 1528.72 1511.69 1510.71 1513.71 1514.71 793.49 776.47 775.48 1430.74 1413.71 1412.73 K[12] D[8] 978.50 961.48 960.49 1006.50 989.47 988.49 1400.62 1383.60 1382.61 1385.61 1386.62 908.52 891.49 890.51 1302.64 1285.62 1284.63 D[11] D[9] 1093.53 1076.50 1075.52 1121.52 1104.50 1103.51 1285.59 1268.57 1267.58 1270.58 1271.59 1023.55 1006.52 1005.54 1187.62 1170.59 1169.61 D[10] F[10] 1240.60 1223.57 1222.59 1268.59 1251.57 1250.58 1170.57 1153.54 1152.56 1155.56 1156.56 1170.62 1153.59 1152.60 1072.59 1055.56 1054.58 F[9] D[11] 1355.62 1338.60 1337.61 1383.62 1366.59 1365.61 1023.50 1006.47 1005.49 1008.49 1009.50 1285.64 1268.62 1267.63 925.52 908.50 907.51 D[8] E[12] 1484.67 1467.64 1466.66 1512.66 1495.64 1494.65 908.47 891.45 890.46 893.46 894.47 1414.68 1397.66 1396.67 810.50 793.47 792.48 E[7] D[13] 1599.69 1582.67 1581.68 1627.69 1610.66 1609.68 779.43 762.40 761.42 764.42 765.43 1529.71 1512.69 1511.70 681.45 664.43 663.44 D[6] Y[14] 1762.76 1745.73 1744.75 1790.75 1773.73 1772.74 664.40 647.38 649.39 650.40 1692.78 1675.75 1674.76 549.40 548.42 Y[5] L[15] 1875.84 1858.81 1857.83 1903.84 1886.81 1885.83 501.34 484.31 486.33 487.34 1805.86 1788.83 1787.85 386.34 385.35 L[4] L[16] 1988.93 1971.90 1970.91 2016.92 1999.89 1998.91 388.26 371.23 373.24 374.25 1918.94 1901.92 1900.93 273.25 272.27 L[3] Q[17] 2116.98 2099.96 2098.97 2144.98 2127.95 2126.97 275.17 258.14 260.16 261.17 2047.00 2029.98 2028.99 160.17 159.18 Q[2] K[18] 2245.08 2228.05 2227.07 2273.07 2256.05 2255.06 147.11 130.09 132.10 133.11 2175.10 2158.07 2157.09 32.11 31.13 K[1]

m/z 500 1000 1500 2000 L I Q Q L S K D D F D E D Y L L Q

SLIDE 20

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590 letter a a* a 0 b b* b 0 y y* y 0 z+1 z+2 b-98 b*-98 b 0-98 y-98 y*-98 y 0-98 letter L[1] 86.10 114.09 2291.08 2274.06 2273.07 2276.07

• 0.91
• 1.90

2193.11 2176.08 2175.10 L[18] I[2] 199.18 227.18 2178.00 2160.97 2159.99 2162.99 112.17 111.19 2080.02 2063.00 2062.01 I[17] Q[3] 327.24 310.21 355.23 338.21 2064.92 2047.89 2046.91 2049.91 2050.91 240.23 239.25 1966.94 1949.91 1948.93 Q[16] Q[4] 455.30 438.27 483.29 466.27 1936.86 1919.83 1918.85 1921.85 1922.85 368.29 367.31 1838.88 1821.85 1820.87 Q[15] L[5] 568.38 551.36 596.38 579.35 1808.80 1791.77 1790.79 1793.79 1794.80 481.37 480.39 1710.82 1693.80 1692.81 L[14] S[6] 735.38 718.35 717.37 763.37 746.35 745.36 1695.71 1678.69 1677.70 1680.70 1681.71 665.40 648.37 647.39 1597.74 1580.71 1579.73 S[13] K[7] 863.48 846.45 845.46 891.47 874.44 873.46 1528.72 1511.69 1510.71 1513.71 1514.71 793.49 776.47 775.48 1430.74 1413.71 1412.73 K[12] D[8] 978.50 961.48 960.49 1006.50 989.47 988.49 1400.62 1383.60 1382.61 1385.61 1386.62 908.52 891.49 890.51 1302.64 1285.62 1284.63 D[11] D[9] 1093.53 1076.50 1075.52 1121.52 1104.50 1103.51 1285.59 1268.57 1267.58 1270.58 1271.59 1023.55 1006.52 1005.54 1187.62 1170.59 1169.61 D[10] F[10] 1240.60 1223.57 1222.59 1268.59 1251.57 1250.58 1170.57 1153.54 1152.56 1155.56 1156.56 1170.62 1153.59 1152.60 1072.59 1055.56 1054.58 F[9] D[11] 1355.62 1338.60 1337.61 1383.62 1366.59 1365.61 1023.50 1006.47 1005.49 1008.49 1009.50 1285.64 1268.62 1267.63 925.52 908.50 907.51 D[8] E[12] 1484.67 1467.64 1466.66 1512.66 1495.64 1494.65 908.47 891.45 890.46 893.46 894.47 1414.68 1397.66 1396.67 810.50 793.47 792.48 E[7] D[13] 1599.69 1582.67 1581.68 1627.69 1610.66 1609.68 779.43 762.40 761.42 764.42 765.43 1529.71 1512.69 1511.70 681.45 664.43 663.44 D[6] Y[14] 1762.76 1745.73 1744.75 1790.75 1773.73 1772.74 664.40 647.38 649.39 650.40 1692.78 1675.75 1674.76 549.40 548.42 Y[5] L[15] 1875.84 1858.81 1857.83 1903.84 1886.81 1885.83 501.34 484.31 486.33 487.34 1805.86 1788.83 1787.85 386.34 385.35 L[4] L[16] 1988.93 1971.90 1970.91 2016.92 1999.89 1998.91 388.26 371.23 373.24 374.25 1918.94 1901.92 1900.93 273.25 272.27 L[3] Q[17] 2116.98 2099.96 2098.97 2144.98 2127.95 2126.97 275.17 258.14 260.16 261.17 2047.00 2029.98 2028.99 160.17 159.18 Q[2] K[18] 2245.08 2228.05 2227.07 2273.07 2256.05 2255.06 147.11 130.09 132.10 133.11 2175.10 2158.07 2157.09 32.11 31.13 K[1]

m/z 500 1000 1500 2000 L I Q Q L S K D D F D E D Y L L Q

SLIDE 21

step 1: computing b- and y-ion matrix

• f LIQQL*SKDDFDEDYLLQK

A 71.037114 B 114.534940 C 160.030649 D 115.026943 E 129.042593 F 147.068414 G 57.021464 H 137.058912 I 113.084064 J 0.000000 K 128.094963 L 113.084064 M 131.040485 N 114.042927 O 0.000000 P 97.052764 Q 128.058578 R 156.101111 S 87.032028 T 101.047679 U 150.953630 V 99.068414 W 186.079313 X 111.000000 Y 163.063329 Z 128.550590 letter a a* a 0 b b* b 0 y y* y 0 z+1 z+2 b-98 b*-98 b 0-98 y-98 y*-98 y 0-98 letter L[1] 86.10 114.09 2291.08 2274.06 2273.07 2276.07

• 0.91
• 1.90

2193.11 2176.08 2175.10 L[18] I[2] 199.18 227.18 2178.00 2160.97 2159.99 2162.99 112.17 111.19 2080.02 2063.00 2062.01 I[17] Q[3] 327.24 310.21 355.23 338.21 2064.92 2047.89 2046.91 2049.91 2050.91 240.23 239.25 1966.94 1949.91 1948.93 Q[16] Q[4] 455.30 438.27 483.29 466.27 1936.86 1919.83 1918.85 1921.85 1922.85 368.29 367.31 1838.88 1821.85 1820.87 Q[15] L[5] 568.38 551.36 596.38 579.35 1808.80 1791.77 1790.79 1793.79 1794.80 481.37 480.39 1710.82 1693.80 1692.81 L[14] S[6] 735.38 718.35 717.37 763.37 746.35 745.36 1695.71 1678.69 1677.70 1680.70 1681.71 665.40 648.37 647.39 1597.74 1580.71 1579.73 S[13] K[7] 863.48 846.45 845.46 891.47 874.44 873.46 1528.72 1511.69 1510.71 1513.71 1514.71 793.49 776.47 775.48 1430.74 1413.71 1412.73 K[12] D[8] 978.50 961.48 960.49 1006.50 989.47 988.49 1400.62 1383.60 1382.61 1385.61 1386.62 908.52 891.49 890.51 1302.64 1285.62 1284.63 D[11] D[9] 1093.53 1076.50 1075.52 1121.52 1104.50 1103.51 1285.59 1268.57 1267.58 1270.58 1271.59 1023.55 1006.52 1005.54 1187.62 1170.59 1169.61 D[10] F[10] 1240.60 1223.57 1222.59 1268.59 1251.57 1250.58 1170.57 1153.54 1152.56 1155.56 1156.56 1170.62 1153.59 1152.60 1072.59 1055.56 1054.58 F[9] D[11] 1355.62 1338.60 1337.61 1383.62 1366.59 1365.61 1023.50 1006.47 1005.49 1008.49 1009.50 1285.64 1268.62 1267.63 925.52 908.50 907.51 D[8] E[12] 1484.67 1467.64 1466.66 1512.66 1495.64 1494.65 908.47 891.45 890.46 893.46 894.47 1414.68 1397.66 1396.67 810.50 793.47 792.48 E[7] D[13] 1599.69 1582.67 1581.68 1627.69 1610.66 1609.68 779.43 762.40 761.42 764.42 765.43 1529.71 1512.69 1511.70 681.45 664.43 663.44 D[6] Y[14] 1762.76 1745.73 1744.75 1790.75 1773.73 1772.74 664.40 647.38 649.39 650.40 1692.78 1675.75 1674.76 549.40 548.42 Y[5] L[15] 1875.84 1858.81 1857.83 1903.84 1886.81 1885.83 501.34 484.31 486.33 487.34 1805.86 1788.83 1787.85 386.34 385.35 L[4] L[16] 1988.93 1971.90 1970.91 2016.92 1999.89 1998.91 388.26 371.23 373.24 374.25 1918.94 1901.92 1900.93 273.25 272.27 L[3] Q[17] 2116.98 2099.96 2098.97 2144.98 2127.95 2126.97 275.17 258.14 260.16 261.17 2047.00 2029.98 2028.99 160.17 159.18 Q[2] K[18] 2245.08 2228.05 2227.07 2273.07 2256.05 2255.06 147.11 130.09 132.10 133.11 2175.10 2158.07 2157.09 32.11 31.13 K[1]

m/z 500 1000 1500 2000 L I Q Q L S K D D F D E D Y L L Q

SLIDE 22 400 600 800 1000 1200 1400 10000 20000 30000 40000 50000 60000 70000

m/z Intensity

SLIDE 23

step 2: setting the ion labels

• bjectives

◮ avoidance of overlapping labels

SLIDE 24

step 2: setting the ion labels

• bjectives

◮ avoidance of overlapping labels

solution heuristic

◮ bins depending on the peptide sequence along the m/z axis ◮ label is only drawn if the corresponding ion count of the m/z

peak is higher than a given threshold

SLIDE 25 400 600 800 1000 1200 1400 10000 20000 30000 40000 50000 60000 70000

m/z Intensity

SLIDE 26 400 600 800 1000 1200 1400 10000 20000 30000 40000 50000 60000 70000

m/z Intensity

• b_0−98(7)2+

c(8)2+ b(4)1+ b_0(8)2+

388.22 512.18 483.41 494.23

y(4)1+ y(2)1+ y*(2)1+ y*(8)2+

501.33 275.13 258.05 503.69

b(11)2+ b(13)2+ b−98(12)2+ b(9)2+

692.25 814.22 707.78 561.25

y*−98(10)2+ y(6)1+ y(5)1+ y*(10)2+

585.79 779.35 664.38 634.75

b(17)2+ b(15)2+ b(16)2+ b(14)2+

1072.85 952.31 1008.83 895.66

y(8)1+ y(7)1+ y_0(15)2+ y*−98(14)2+

1023.44 908.38 959.86 847.2

b(10)1+

1268.4

y(9)1+ y*−98(11)1+

1170.46 1285.52 title: peakplot query number: q1181_p1 mascot Score: 80.54 precursormass/charge: 764.366760,3+ delta score; pep rank1 − pep rank2: 56.38 peptide sequence: LIQQL*SKDDFDEDYLLQK

[M+3H]−98

y−98(13)++ y(13)++

SLIDE 27 400 600 800 1000 1200 1400 10000 20000 30000 40000 50000 60000 70000

m/z Intensity

• b_0−98(7)2+

c(8)2+ b(4)1+ b_0(8)2+

388.22 512.18 483.41 494.23

y(4)1+ y(2)1+ y*(2)1+ y*(8)2+

501.33 275.13 258.05 503.69

b(11)2+ b(13)2+ b−98(12)2+ b(9)2+

692.25 814.22 707.78 561.25

y*−98(10)2+ y(6)1+ y(5)1+ y*(10)2+

585.79 779.35 664.38 634.75

b(17)2+ b(15)2+ b(16)2+ b(14)2+

1072.85 952.31 1008.83 895.66

y(8)1+ y(7)1+ y_0(15)2+ y*−98(14)2+

1023.44 908.38 959.86 847.2

b(10)1+

1268.4

y(9)1+ y*−98(11)1+

1170.46 1285.52 title: peakplot query number: q1181_p1 mascot Score: 80.54 precursormass/charge: 764.366760,3+ delta score; pep rank1 − pep rank2: 56.38 peptide sequence: LIQQL*SKDDFDEDYLLQK

[M+3H]−98

y−98(13)++ y(13)++

SLIDE 28

800

m/z

• y−98(13)++

y(13)++

SLIDE 29

800

m/z

• y−98(13)++

y(13)++

• 1200
• y(9)1+

1170.46

SLIDE 30 a(10) a(12) a(14) a(15) a(16) b(3) b(4) b(5) b(8) b(9) b(10) b(11) b(13) b(14) b(15) b(16) b(17) y(2) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(13) y(16) rest

• 400

600 800 1000 1200 1400 0.0 0.1 0.2 0.3 0.4 0.5

m/z abs error

a(10)+ a(12)+ a(14)+ a(15)+ a(16)+ a*(6)+ a*(10)+ a_0(12)+ a_0(14)+ a_0(15)+ b(3)+ b(4)+ b(5)+ b(8)+ b(9)+ b(10)+ b(11)+ b(13)+ b(14)+ b(15)+ b(16)+ b(17)+ b*(5)+ b*(10)+ b*(13)+ b*−98(10)+ b*−98(12)+ b−98(8)+ b−98(9)+ b−98(12)+ b−98(15)+ b_0(6)+ b_0(8)+ b_0(10)+ b_0(11)+ b_0(12)+ b_0(13)+ b_0(14)+ b_0(15)+ b_0(16)+ b_0(17)+ b_0−98(7)+ b_0−98(8)+ b_0−98(11)+ b_0−98(17)+ c(8)+ c(13)+ y(2)+ y(4)+ y(5)+ y(6)+ y(7)+ y(8)+ y(9)+ y(10)+ y(13)+ y(16)+ y*(2)+ y*(3)+ y*(4)+ y*(5)+ y*(8)+ y*(9)+ y*(10)+ y*(11)+ y*−98(10)+ y*−98(11)+ y*−98(14)+ y*−98(16)+ y−98(6)+ y−98(12)+ y−98(13)+ y−98(14)+ y−98(15)+ y−98(16)+ y_0(7)+ y_0(13)+ y_0(15)+ y_0(16)+ y_0−98(8)+ z+1(3)+ z+1(8)+

SLIDE 31 500 1000 1500 100 200 300 400 500

m/z Intensity

• b(6)1+

b_0(6)1+ b(5)1+ 653.48 635.13 552.36 y*−98(10)2+ y(4)1+ y(3)1+ y(6)2+ 564.39 620.32 457.17 408.11 b(9)1+ b(8)1+ b_0(9)1+ b(7)1+ 994.44 895.52 976.45 766.48 y(5)1+ y(7)1+ y(16)2+ y*(16)2+ 717.42 913.41 961.99 953.46 b_0(20)2+ b(10)1+ b(11)1+ b_0(11)1+ 1150.42 1095.54 1208.64 1190.51 y(9)1+ y(8)1+ y(18)2+ y(10)1+ 1129.58 1042.47 1076.94 1242.6 b_0(13)1+ b(13)1+ b(12)1+ 1406.58 1424.67 1295.69 y(11)1+ y(13)1+ y(12)1+ 1343.51 1571.77 1442.68 b(15)1+ b_0−98(16)1+ 1620.71 1602.12 y(14)1+ y(15)1+ 1684.78 1785.96 title: peakplot query number: q10968_p1 mascot Score: 116.99 precursormass/charge: peptide sequence: AIDDHTLEVTLSEPVPYFYK

SLIDE 32 500 1000 1500 50 100 150 200 250 300

m/z Intensity

• b(11)2+

b−98(15)2+ 382.13 530.46 y(8)1+ y(7)1+ y_0−98(9)1+ y(5)1+ 574.37 517.56 545.36 403.26 b_0(9)1+ b_0(11)1+ a_0(9)1+ b*−98(21)2+ 632.4 746.45 603.98 776.47 y−98(19)2+ y(9)1+ y_0(9)1+ y_0(20)2+ 718.48 661.44 643.68 801.26 b_0(13)1+ b_0(12)1+ a_0(25)2+ b_0−98(27)2+ 920.34 833.44 940.49 961.22 y(11)1+ y(12)1+ y(25)2+ y*(25)2+ 881.47 968.5 1021.25 1012.76 b_0(31)2+ b_0(14)1+ b(14)1+ b(28)2+ 1173.98 1083.56 1101.52 1048.29 y(15)1+ y(14)1+ y(16)1+ y*(14)1+ 1139.58 1082.47 1226.61 1065.33 b(18)1+ b(19)1+ 1359.57 1416.48 y(17)1+ y(18)1+ y_0(17)1+ y*(18)1+ 1283.6 1446.65 1265.42 1429.37 a_0(21)1+ b_0(21)1+ b_0(20)1+ b(20)1+ 1620.57 1648.59 1485.49 1503.59 y(19)1+ y_0(19)1+ y_0(21)1+ 1533.76 1515.88 1659.68 b(21)1+ b_0(24)1+ b_0(23)1+ 1666.29 1849.76 1791.98 y(22)1+ y(21)1+ y−98(23)1+ 1734.7 1677.83 1723.81 title: peakplot query number: q11155_p1 mascot Score: 115.35 precursormass/charge: 1192.481100,2+ delta score; pep rank1 − pep rank2: 105.54 peptide sequence: GGGGGGYGSGGSSYGSGGGSYGSGGGGGGGR [M+2H]−98

SLIDE 33 500 1000 1500 100 200 300 400 500 600

m/z Intensity

• b(4)1+

474.56 y(5)1+ y(4)1+ y(3)1+ y_0(5)1+ 602.33 473.19 359.42 584.38 b(7)1+ b*−98(18)2+ b*(8)1+ b*(7)1+ 731.37 844.42 827.38 714.32 y(7)1+ 832.53 b*(20)2+ b_0(19)2+ a*(21)2+ b_0(10)1+ 1015.17 957.38 1044.3 1026.46 y(9)1+ y(21)2+ y*(20)2+ y(8)1+ 1020.47 1071.75 1006.01 933.48 b*(23)2+ b(12)1+ b(14)1+ c(12)1+ 1185.15 1158.37 1384.48 1176.31 y(13)1+ y(11)1+ y(12)1+ y*(13)1+ 1360.62 1246.63 1303.58 1343.85 b_0(17)1+ b(16)1+ b_0(16)1+ 1655.86 1572.72 1554.16 y(14)1+ y(15)1+ y_0(14)1+ y_0(15)1+ 1447.7 1560.79 1429.72 1542.87 title: peakplot query number: q11272_p1 mascot Score: 130.31 precursormass/charge: 1202.568600,2+ delta score; pep rank1 − pep rank2: 129.28 peptide sequence: DFNPSGIILSGGPESTTEENSPR [M+2H]−98

SLIDE 34 500 1000 1500 2000 100 200 300 400 500 600

m/z Intensity

• b*−98(6)1+

b_0(6)1+ b*(14)2+ b(4)1+ 570.43 667.57 740.15 457.22 y(5)1+ y(4)1+ z+2(13)2+ z+1(12)2+ 622.14 459.46 566.44 614.7 b(7)1+ a(7)1+ b(9)1+ b_0(10)1+ 813.32 785.55 1015.56 1068.46 y(7)1+ y*(16)2+ y_0−98(18)2+ y(15)2+ 948.46 928.58 999.5 879.67 b(12)1+ b(11)1+ b*(21)2+ b*−98(13)1+ 1298.6 1199.54 1203.92 1280.73 y(9)1+ y(11)1+ y*(19)2+ y(10)1+ 1144.57 1356.63 1113.23 1243.5 b(14)1+ b(15)1+ 1494.83 1657.79 y(13)1+ y(14)1+ y(12)1+ y_0(14)1+ 1514.78 1629.2 1427.8 1611.18 title: peakplot query number: q11426_p1 mascot Score: 117.52 precursormass/charge: 1221.621700,2+ delta score; pep rank1 − pep rank2: 116.67 peptide sequence: AEQQLDKDSAIVPVYYYVNAR [M+2H]−98

SLIDE 35 400 600 800 1000 1200 1400 100 200 300 400 500 600 700

m/z Intensity

• b_0(5)1+

b(5)1+ 482.22 500.42 y(8)2+ y_0−98(5)1+ y(9)2+ y(3)1+ 466.5 472.41 523.91 347.27 b(7)1+ b_0(6)1+ a(14)2+ b_0−98(13)2+ 684.49 595.11 697.19 624.4 y*−98(6)1+ y*−98(7)1+ y*−98(12)2+ y_0−98(7)1+ 588.57 703.59 587.9 702.64 a(8)1+ b(8)1+ a(15)2+ c(8)1+ 770.18 798.1 733.13 814.87 z+2(18)1+ y*−98(16)2+ y(7)1+ y_0(17)2+ 804.63 813.98 818.53 918.9 b*−98(11)1+ b(19)2+ a(21)2+ a(11)1+ 1020.37 975.9 1082.05 1108.35 y(18)2+ y_0(10)1+ y(21)2+ y(17)2+ 963.73 1099.76 1099.06 928.29 b(22)2+ b(11)1+ b(23)2+ b_0(23)2+ 1145.89 1135.53 1197.01 1187.82 y(22)2+ y*(22)2+ y(11)1+ y(23)2+ 1163.9 1154.96 1174.63 1220.27 title: peakplot query number: q11762_p1 mascot Score: 121.58 precursormass/charge: 846.761800,3+ delta score; pep rank1 − pep rank2: 121.37 peptide sequence: VLESAIANAEHNDGADIDDLKVTK [M+3H]−98

SLIDE 36 a(10) a(17) a(19) a(25) b(6) b(8) b(9) b(12) y(4) y(5) y(9) y(11) y(12) y(13) y(14) y(16) y(17) y(18) y(19) y(22) y(23) y(24) y(25) y(26) y(27) y(28) rest a(12) a(28) b(3) b(8) b(11) b(21) b(27) b(31) y(4) y(7) y(8) y(9) y(12) y(13) y(15) y(16) y(17) y(18) y(19) y(20) y(21) y(22) y(23) y(24) y(26) y(27) y(28) y(29) y(31) rest a(5) b(3) b(4) b(5) b(7) b(10) b(11) b(12) b(15) b(16) b(18) y(4) y(5) y(6) y(7) y(8) y(10) y(11) y(12) y(13) y(14) y(15) y(16) y(17) y(18) rest b(5) b(6) b(9) b(24) b(27) b(31) y(6) y(7) y(11) y(13) y(14) y(16) y(17) y(18) y(19) y(21) y(23) y(25) y(27) y(28) y(29) y(31) rest a(7) a(13) b(3) b(6) b(7) b(9) b(16) b(19) b(20) y(5) y(6) y(10) y(11) y(12) y(13) rest a(22) b(4) b(7) b(12) b(14) b(16) b(17) b(18) y(3) y(4) y(5) y(7) y(8) y(9) y(11) y(12) y(13) y(14) y(15) y(19) y(21) rest a(9) a(11) a(16) b(4) b(6) b(9) b(10) b(15) b(16) y(1) y(2) y(3) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(14) y(15) rest a(16) b(5) b(6) b(8) b(9) b(10) b(17) b(19) b(20) b(22) b(24) b(25) b(27) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(15) y(16) y(17) y(18) y(19) y(20) y(22) y(23) y(24) y(25) y(26) y(27) rest b(8) b(9) b(11) b(14) b(15) y(5) y(6) y(7) y(10) y(15) y(16) y(18) y(19) y(21) y(23) y(25) y(26) y(27) y(28) y(29) y(31) rest b(3) b(4) b(5) b(7) b(9) b(10) b(11) b(12) b(13) b(14) y(2) y(3) y(4) y(7) y(9) y(10) y(11) y(12) y(13) y(14) rest a(8) a(11) a(14) a(15) a(21) a(22) b(5) b(7) b(8) b(11) b(13) b(15) b(19) b(22) b(23) y(3) y(7) y(8) y(9) y(10) y(11) y(12) y(15) y(16) y(17) y(18) y(20) y(21) y(22) y(23) rest a(8) a(9) b(4) b(6) b(7) b(8) b(9) b(10) b(11) b(12) b(13) b(15) b(16) b(17) y(4) y(5) y(6) y(7) y(9) y(10) y(11) y(12) y(13) y(14) y(15) y(16) y(17) rest a(6) a(7) a(8) a(11) a(13) b(4) b(5) b(8) b(9) b(10) b(11) b(13) b(15) y(2) y(3) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(14) y(15) rest a(8) a(9) a(16) b(3) b(4) b(5) b(6) b(8) b(13) b(14) b(16) b(17) y(2) y(4) y(5) y(7) y(9) y(10) y(11) y(12) y(13) y(14) y(16) rest a(15) a(18) a(19) a(20) b(6) b(7) b(9) b(10) b(11) b(12) b(13) b(15) b(17) b(21) b(23) b(24) b(27) y(3) y(4) y(5) y(6) y(8) y(9) y(10) y(11) y(12) y(14) y(16) y(17) y(19) y(22) rest a(9) a(16) a(17) b(8) b(9) b(12) b(13) b(15) b(17) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(15) y(16) y(18) rest

SLIDE 37 a(10) a(17) a(19) a(25) b(6) b(8) b(9) b(12) y(4) y(5) y(9) y(11) y(12) y(13) y(14) y(16) y(17) y(18) y(19) y(22) y(23) y(24) y(25) y(26) y(27) y(28) rest a(12) a(28) b(3) b(8) b(11) b(21) b(27) b(31) y(4) y(7) y(8) y(9) y(12) y(13) y(15) y(16) y(17) y(18) y(19) y(20) y(21) y(22) y(23) y(24) y(26) y(27) y(28) y(29) y(31) rest a(5) b(3) b(4) b(5) b(7) b(10) b(11) b(12) b(15) b(16) b(18) y(4) y(5) y(6) y(7) y(8) y(10) y(11) y(12) y(13) y(14) y(15) y(16) y(17) y(18) rest b(5) b(6) b(9) b(24) b(27) b(31) y(6) y(7) y(11) y(13) y(14) y(16) y(17) y(18) y(19) y(21) y(23) y(25) y(27) y(28) y(29) y(31) rest a(7) a(13) b(3) b(6) b(7) b(9) b(16) b(19) b(20) y(5) y(6) y(10) y(11) y(12) y(13) rest a(22) b(4) b(7) b(12) b(14) b(16) b(17) b(18) y(3) y(4) y(5) y(7) y(8) y(9) y(11) y(12) y(13) y(14) y(15) y(19) y(21) rest a(9) a(11) a(16) b(4) b(6) b(9) b(10) b(15) b(16) y(1) y(2) y(3) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(14) y(15) rest a(16) b(5) b(6) b(8) b(9) b(10) b(17) b(19) b(20) b(22) b(24) b(25) b(27) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(15) y(16) y(17) y(18) y(19) y(20) y(22) y(23) y(24) y(25) y(26) y(27) rest b(8) b(9) b(11) b(14) b(15) y(5) y(6) y(7) y(10) y(15) y(16) y(18) y(19) y(21) y(23) y(25) y(26) y(27) y(28) y(29) y(31) rest b(3) b(4) b(5) b(7) b(9) b(10) b(11) b(12) b(13) b(14) y(2) y(3) y(4) y(7) y(9) y(10) y(11) y(12) y(13) y(14) rest a(8) a(11) a(14) a(15) a(21) a(22) b(5) b(7) b(8) b(11) b(13) b(15) b(19) b(22) b(23) y(3) y(7) y(8) y(9) y(10) y(11) y(12) y(15) y(16) y(17) y(18) y(20) y(21) y(22) y(23) rest a(8) a(9) b(4) b(6) b(7) b(8) b(9) b(10) b(11) b(12) b(13) b(15) b(16) b(17) y(4) y(5) y(6) y(7) y(9) y(10) y(11) y(12) y(13) y(14) y(15) y(16) y(17) rest a(6) a(7) a(8) a(11) a(13) b(4) b(5) b(8) b(9) b(10) b(11) b(13) b(15) y(2) y(3) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(14) y(15) rest a(8) a(9) a(16) b(3) b(4) b(5) b(6) b(8) b(13) b(14) b(16) b(17) y(2) y(4) y(5) y(7) y(9) y(10) y(11) y(12) y(13) y(14) y(16) rest a(15) a(18) a(19) a(20) b(6) b(7) b(9) b(10) b(11) b(12) b(13) b(15) b(17) b(21) b(23) b(24) b(27) y(3) y(4) y(5) y(6) y(8) y(9) y(10) y(11) y(12) y(14) y(16) y(17) y(19) y(22) rest a(9) a(16) a(17) b(8) b(9) b(12) b(13) b(15) b(17) y(4) y(5) y(6) y(7) y(8) y(9) y(10) y(11) y(12) y(13) y(15) y(16) y(18) rest

SLIDE 38 600 800 1000 1200 1400 1600 1800 500 1000 1500 m/z Intensity

• b*(6)1+

b(6)1+ 601.34 617.82 y−98(11)2+ y(5)1+ y_0−98(6)1+ z+2(27)1+ 571.64 581.53 621.21 567.25 b*(7)1+ b−98(17)2+ b_0(7)1+ 729.43 746.41 728.36 y(12)2+ y(13)2+ y*−98(7)1+ y(14)2+ 649.75 706.99 737.45 763.88 b_0(8)1+ b(8)1+ b*(8)1+ a(17)2+ 799.52 817.52 800.44 781.38 y(17)2+ y(16)2+ z+1(14)2+ y_0−98(16)2+ 856.3 827.92 899.15 769.47 b*(10)1+ b_0(9)1+ b_0(20)2+ c(21)2+ 1014.42 912.44 928.27 995.28 y(19)2+ y−98(9)1+ y*−98(10)1+ y(18)2+ 963.43 957.29 1027.05 906.7 b_0(12)1+ b*(11)1+ 1171.27 1071.28 y(22)2+ y(23)2+ y*(22)2+ y*(23)2+ 1093.45 1149.73 1084.48 1140.83 a(25)2+ b(12)1+ 1175.65 1189.65 y(24)2+ y(25)2+ y*−98(13)1+ y(26)2+ 1185.52 1249.45 1297.75 1299.88 title: peakplot query number: q13533_p1 mascot Score: 157.11 precursormass/charge: peptide sequence: DLSSNTQALTGTNTGGAIDGVSTTDRSPYSK 400 600 800 1000 1200 1400 1600 50 100 150 200 250 300 m/z Intensity

• b(3)1+

a*(4)1+ b_0(3)1+ 375.32 443.41 357.38 y(4)1+ 494.33 a(12)2+ c(5)1+ 632.12 592.36 y(12)2+ y−98(11)2+ y_0(11)2+ 649.68 572 611.92 b*(8)1+ b(8)1+ b_0(8)1+ 860.4 877.38 859.41 y*−98(7)1+ y(17)2+ y(16)2+ y(7)1+ 737.5 856.5 827.96 852.47 b(21)2+ a*(10)1+ b_0(9)1+ b*(19)2+ 1039.07 1031.58 987.92 917.06 y(21)2+ y(19)2+ y(18)2+ y_0(20)2+ 1042.81 963.82 906.79 1004.34 b_0(23)2+ b(11)1+ c(22)2+ b_0(11)1+ 1108.08 1189.45 1076.21 1172.02 y(22)2+ y(24)2+ y*(22)2+ z+1(9)2+ 1093.08 1185.35 1084.37 1241.44 b(27)2+ b_0(12)1+ a(28)2+ b*(27)2+ 1319.29 1272.5 1383 1310.53 y(28)2+ y*−98(13)1+ y(26)2+ y(27)2+ 1400.08 1297.65 1300.36 1357.11 b*−98(16)1+ b(31)2+ b_0−98(17)1+ 1548.84 1571.35 1604.97 y(31)2+ y(29)2+ y*−98(31)2+ y*(29)2+ 1558 1444.2 1500.61 1435.17 title: peakplot query number: q13927_p1 mascot Score: 136.36 precursormass/charge: 1125.212400,3+ delta score; pep rank1 − pep rank2: 135.8 peptide sequence: MKDLSSNTQALTGTNTGGAIDGVSTTDRSPYSK [M+3H]−98 500 1000 1500 200 400 600 800 1000 1200 1400 m/z Intensity

• b(5)1+

a_0(10)2+ a(5)1+ b(4)1+ 567.44 454.5 539.51 438.18 y(4)1+ y*−98(4)1+ y(5)1+ y_0−98(11)2+ 502.47 387.49 630.36 549.4 b_0(7)1+ b(7)1+ b*−98(10)1+ b_0(9)1+ 707.42 725.42 839.44 821.49 y(7)1+ y_0−98(9)1+ y*−98(18)2+ y−98(18)2+ 814.51 925.56 884 893.19 b(12)1+ b_0(12)1+ b(10)1+ b*(19)2+ 1181.58 1163.49 954.47 980.4 y(8)1+ y*−98(10)1+ y(18)2+ y(11)1+ 942.65 1041.63 941.83 1213.61 b(15)1+ b_0(14)1+ 1493.43 1347.65 y(14)1+ y(12)1+ y(13)1+ y_0(14)1+ 1428.67 1270.56 1371.71 1410.71 b(16)1+ b_0(18)1+ 1608.47 1802.82 y(15)1+ y(16)1+ y_0(15)1+ y*(17)1+ 1557.83 1656.87 1539.68 1767.83 title: peakplot query number: q9424_p1 mascot Score: 135.93 precursormass/charge: peptide sequence: LPQVEGTGGDVQPSQDLVR 600 800 1000 1200 1400 1600 50 100 150 200 250 300 350 m/z Intensity

• b(6)1+

b_0(6)1+ b_0−98(7)1+ b(5)1+ 587.27 569.5 586.05 458.54 y(11)2+ y−98(10)2+ y*−98(11)2+ 609.54 502.49 551.94 b_0−98(8)1+ c(15)2+ b*(7)1+ 698.84 780.46 685.26 y(14)2+ y*(6)1+ z+1(27)1+ y−98(14)2+ 753.08 629.01 744.68 704.47 b_0−98(17)2+ b_0(8)1+ b*−98(18)2+ b(9)1+ 798.46 797.47 834.78 930.17 y(17)2+ y(16)2+ y(18)2+ y*−98(16)2+ 919.68 870 955.5 812.55 b_0−98(12)1+ c(21)2+ b*(10)1+ b_0(11)1+ 1125.67 1082.9 1026.52 1124.51 y(21)2+ y*−98(21)2+ y(19)2+ y−98(21)2+ 1104.87 1048 1012.33 1056.3 b(24)2+ b_0(12)1+ 1224.47 1223.09 y(23)2+ y*−98(25)2+ 1204.53 1260.69 b(27)2+ b*(26)2+ 1402.09 1337.8 y(25)2+ y(27)2+ y_0−98(27)2+ y*(25)2+ 1318.45 1432.24 1374.27 1309.9 b(31)2+ 1596.16 y_0(14)1+ y_0−98(30)2+ y(28)2+ y(29)2+ 1486.99 1538.63 1496.2 1540.45 title: peakplot query number: q14020_p1 mascot Score: 133.57 precursormass/charge: 1150.582900,3+ delta score; pep rank1 − pep rank2: 132.11 peptide sequence: SVANSQDIDLVVDANAVAYNSSDVKDITADVLK [M+3H]−98 500 1000 1500 100 200 300 400 500 600 m/z Intensity

• a(7)2+

b*(5)2+ c(6)2+ b_0(6)2+ 363.24 284.09 357.43 339.24 y−98(8)2+ y_0−98(10)2+ y*(6)2+ y_0−98(8)2+ 419.49 488.5 367.52 410.92 a*(7)1+ b*(6)1+ c(12)2+ b−98(7)1+ 708.73 679.44 649.49 655.78 z+1(9)2+ y_0(12)2+ y(5)1+ y*−98(5)1+ 695.58 629.97 665.01 550.13 b(7)1+ b*−98(8)1+ a*(15)2+ b*(17)2+ 754.16 793.94 760.27 845.98 y(6)1+ z+2(16)1+ y_0(8)1+ y*−98(18)2+ 750.77 737.03 919.18 929.6 b(9)1+ b(19)2+ b_0−98(20)2+ a_0(21)2+ 1037.45 997.51 1012.05 1125.32 y_0(9)1+ z+1(4)2+ y−98(11)1+ y_0−98(19)2+ 1016.25 979.2 1093.33 985.14 b*(11)1+ a*(12)1+ 1206.64 1235.72 y(11)1+ 1190.2 title: peakplot query number: q10882_p1 mascot Score: 0.02 precursormass/charge: peptide sequence: DIIKLLGRQSVGPDASGWVFR 500 1000 1500 100 200 300 400 500 600 m/z Intensity

• b(4)1+

474.56 y(5)1+ y(4)1+ y(3)1+ y_0(5)1+ 602.33 473.19 359.42 584.38 b(7)1+ b*−98(18)2+ b*(8)1+ b*(7)1+ 731.37 844.42 827.38 714.32 y(7)1+ 832.53 b*(20)2+ b_0(19)2+ a*(21)2+ b_0(10)1+ 1015.17 957.38 1044.3 1026.46 y(9)1+ y(21)2+ y*(20)2+ y(8)1+ 1020.47 1071.75 1006.01 933.48 b*(23)2+ b(12)1+ b(14)1+ c(12)1+ 1185.15 1158.37 1384.48 1176.31 y(13)1+ y(11)1+ y(12)1+ y*(13)1+ 1360.62 1246.63 1303.58 1343.85 b_0(17)1+ b(16)1+ b_0(16)1+ 1655.86 1572.72 1554.16 y(14)1+ y(15)1+ y_0(14)1+ y_0(15)1+ 1447.7 1560.79 1429.72 1542.87 title: peakplot query number: q11272_p1 mascot Score: 130.31 precursormass/charge: 1202.568600,2+ delta score; pep rank1 − pep rank2: 129.28 peptide sequence: DFNPSGIILSGGPESTTEENSPR [M+2H]−98 200 400 600 800 1000 500 1000 1500 2000 m/z Intensity

• b_0(6)1+

b_0(5)1+ b(6)1+ a(11)2+ 355.23 298.15 373.12 359.35 z+1(8)2+ y(3)1+ y(2)1+ y(1)1+ 394.28 375.16 288.28 175.18 b(16)2+ b_0(9)1+ b_0(16)2+ b(9)1+ 607.94 582.34 598.89 600.31 y(5)1+ y(6)1+ y_0(15)2+ y(14)2+ 489.45 576.36 564.21 544.9 b_0(10)1+ b_0(11)1+ b_0(12)1+ b(10)1+ 639.35 726.44 783.24 657.32 y(7)1+ y(9)1+ y(8)1+ y_0−98(10)1+ 633.42 803.49 746.48 744.4 b_0(15)1+ b_0(14)1+ b_0(13)1+ 1040.36 927.37 840.29 y(10)1+ y(11)1+ y(12)1+ y_0(10)1+ 860.6 917.48 974.57 842.69 title: peakplot query number: q4437_p1 mascot Score: 126.36 precursormass/charge: peptide sequence: SGGGGGGGLGSGGSIR 400 600 800 1000 1200 1400 1600 100 200 300 400 500 m/z Intensity

• b(5)1+

600.36 y(6)1+ 619.4 b*(6)1+ b(6)1+ b*(7)1+ 746.47 763.25 875.02 y(8)1+ y(7)1+ y(16)2+ y(15)2+ 876.4 748.59 845.07 780.47 a_0(17)2+ b(17)2+ a_0(18)2+ b_0(17)2+ 1013.01 1035.98 1056.45 1027.03 y(19)2+ y(17)2+ y_0(19)2+ y(18)2+ 1016.13 901.27 1006.88 965.71 b_0(18)2+ b_0−98(21)2+ b(20)2+ b_0(20)2+ 1070.54 1136.9 1166.2 1157.62 y(12)1+ y(22)2+ y(20)2+ y(11)1+ 1194.53 1186.93 1072.73 1106.92 b(10)1+ b_0(24)2+ b(24)2+ b(22)2+ 1234.4 1363.67 1373.64 1259.21 y(24)2+ y(23)2+ y_0(25)2+ y_0(24)2+ 1333.4 1251.69 1388.13 1323.9 b(25)2+ b_0(25)2+ b_0(28)2+ b(27)2+ 1437.22 1428.29 1550.19 1523.67 y(26)2+ y(27)2+ y(25)2+ y*(26)2+ 1453.72 1518.25 1397.26 1445.69 title: peakplot query number: q13741_p1 mascot Score: 125.36 precursormass/charge: 1088.846000,3+ delta score; pep rank1 − pep rank2: 119.84 peptide sequence: DIENQYETQITQIEHEVSSSGQEVQSSAK [M+3H]−98 600 800 1000 1200 1400 1600 1800 200 400 600 800 1000 1200 1400 m/z Intensity

• a*(7)1+

626.21 y(5)1+ y(6)1+ 515.47 628.66 b(14)2+ b(8)1+ 752.85 800.54 y(18)2+ y(16)2+ y(7)1+ 900.97 803.13 765.6 b_0(11)1+ b*−98(21)2+ b(9)1+ b_0−98(23)2+ 1095.53 1006.15 913.43 1090.6 y(21)2+ y(19)2+ y*−98(21)2+ y(10)1+ 1050.34 936.61 993.21 1080.47 c(22)2+ b(11)1+ a*(26)2+ b*(12)1+ 1107.64 1113.64 1283.56 1259.4 y_0(12)1+ y(25)2+ y(23)2+ y_0(25)2+ 1232.58 1289.02 1188.43 1279.32 b−98(29)2+ 1430.42 y(28)2+ y(27)2+ y(29)2+ y*(26)2+ 1425.46 1396.91 1468.92 1344.97 b−98(31)2+ b(15)1+ b_0(15)1+ b*−98(31)2+ 1516.97 1575.62 1557.71 1508.44 y(31)2+ y(15)1+ 1573.2 1503.75 title: peakplot query number: q13931_p1 mascot Score: 125.15 precursormass/charge: 1125.942100,3+ delta score; pep rank1 − pep rank2: 122.95 peptide sequence: TQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVK [M+3H]−98 400 600 800 1000 1200 1400 1000 2000 3000 4000 m/z Intensity

• b_0(5)1+

b(5)1+ a*(9)2+ b(4)1+ 545.3 563.29 444.31 450.22 y(4)1+ y(3)1+ y(2)1+ z+2(8)2+ 556.42 409.44 322.15 527.19 b_0(16)2+ b*−98(8)1+ b_0(6)1+ b_0(8)1+ 876.48 703.45 646.41 800.46 y(7)1+ y*−98(6)1+ y(14)2+ y_0(14)2+ 857.39 685.48 786.98 777.97 b_0(9)1+ b_0(10)1+ b(11)1+ b(9)1+ 913.43 970.54 1103.55 931.43 y(10)1+ y(9)1+ y_0(10)1+ y_0(8)1+ 1124.58 1067.53 1106.58 952.42 b_0(13)1+ b(13)1+ b(12)1+ 1361.53 1379.49 1232.54 y(11)1+ y(12)1+ y(13)1+ y_0(11)1+ 1225.57 1338.73 1425.8 1207.86 title: peakplot query number: q8296_p1 mascot Score: 122.81 precursormass/charge: 894.436660,2+ delta score; pep rank1 − pep rank2: 117.22 peptide sequence: TNFSLTGPLGDEFSFR [M+2H]−98 400 600 800 1000 1200 1400 100 200 300 400 500 600 700 m/z Intensity

• b_0(5)1+

b(5)1+ 482.22 500.42 y(8)2+ y_0−98(5)1+ y(9)2+ y(3)1+ 466.5 472.41 523.91 347.27 b(7)1+ b_0(6)1+ a(14)2+ b_0−98(13)2+ 684.49 595.11 697.19 624.4 y*−98(6)1+ y*−98(7)1+ y*−98(12)2+ y_0−98(7)1+ 588.57 703.59 587.9 702.64 a(8)1+ b(8)1+ a(15)2+ c(8)1+ 770.18 798.1 733.13 814.87 z+2(18)1+ y*−98(16)2+ y(7)1+ y_0(17)2+ 804.63 813.98 818.53 918.9 b*−98(11)1+ b(19)2+ a(21)2+ a(11)1+ 1020.37 975.9 1082.05 1108.35 y(18)2+ y_0(10)1+ y(21)2+ y(17)2+ 963.73 1099.76 1099.06 928.29 b(22)2+ b(11)1+ b(23)2+ b_0(23)2+ 1145.89 1135.53 1197.01 1187.82 y(22)2+ y*(22)2+ y(11)1+ y(23)2+ 1163.9 1154.96 1174.63 1220.27 title: peakplot query number: q11762_p1 mascot Score: 121.58 precursormass/charge: 846.761800,3+ delta score; pep rank1 − pep rank2: 121.37 peptide sequence: VLESAIANAEHNDGADIDDLKVTK [M+3H]−98 500 1000 1500 200 400 600 800 m/z Intensity

• b(10)2+

b*−98(11)2+ b*(5)1+ a*(11)2+ 577.27 569.51 559.85 604.08 y(6)1+ y(5)1+ y(4)1+ 608.38 445.28 374.33 b(6)1+ b(7)1+ b*(6)1+ b*(7)1+ 690.29 789.52 673.32 772.37 y(9)1+ y(7)1+ y−98(16)2+ y(16)2+ 877.55 679.39 778.62 827.84 b_0(19)2+ b(8)1+ b(9)1+ b_0(8)1+ 997.6 926.32 1054.42 908.48 y(10)1+ y(11)1+ y*−98(19)2+ z+2(10)1+ 976.54 1104.82 958.27 962.93 b(11)1+ b(12)1+ b(13)1+ b*−98(13)1+ 1252.54 1351.7 1422.5 1307.34 y(12)1+ y(13)1+ y*(14)1+ y_0−98(13)1+ 1241.73 1340.68 1437.23 1224.78 b(15)1+ 1656.52 y(14)1+ y(15)1+ 1453.83 1524.75 title: peakplot query number: q9664_p1 mascot Score: 120.70 precursormass/charge: peptide sequence: DFNEALVHQVVVAYAAGAR 400 600 800 1000 1200 1400 100 200 300 400 m/z Intensity

• b*(5)1+

b(5)1+ b_0−98(6)1+ a_0(12)2+ 495.07 512.42 525.29 569.46 y(5)1+ y(4)1+ y_0(5)1+ y(3)1+ 560.5 473.35 542.63 359.35 b(8)1+ b*(8)1+ b*(7)1+ b_0(8)1+ 811.33 794.38 695.31 793.72 y(8)1+ y(7)1+ y(6)1+ y*(14)2+ 845.52 730.43 659.48 713.89 b(11)1+ b_0(11)1+ b(9)1+ b(10)1+ 1096.39 1078.43 926.3 997.36 y(10)1+ y(9)1+ y(11)1+ 1015.61 944.61 1144.55 b_0(15)1+ a_0(13)1+ b(13)1+ a(13)1+ 1463.52 1251.84 1297.47 1269.47 y(12)1+ y(14)1+ y*(12)1+ 1258.65 1443.64 1241.85 title: peakplot query number: q7520_p1 mascot Score: 120.20 precursormass/charge: 828.440230,2+ delta score; pep rank1 − pep rank2: 118.78 peptide sequence: VIGQNEAVDAVSNAIR [M+2H]−98 400 600 800 1000 1200 1400 1600 1000 2000 3000 4000 m/z Intensity

• b_0(6)1+

b(5)1+ b(4)1+ b*(6)1+ 622.43 527.39 428.32 623.35 y(5)1+ y(4)1+ y−98(14)2+ y(2)1+ 551.46 464.44 619.72 322.4 b(8)1+ b_0(9)1+ b_0(8)1+ b(6)1+ 768.47 807.47 750.48 640.39 y(9)1+ y*(8)1+ y(7)1+ y_0(17)2+ 939.5 825.37 771.56 822.47 b(13)1+ b_0(11)1+ 1213.58 975.49 y(10)1+ y(12)1+ y(13)1+ y(11)1+ 996.55 1124.57 1237.7 1067.5 b(16)1+ b(14)1+ 1442.72 1300.5 y(14)1+ 1336.74 title: peakplot query number: q8182_p1 mascot Score: 120.06 precursormass/charge: 882.477120,2+ delta score; pep rank1 − pep rank2: 104.99 peptide sequence: TQVVVLGAGPAGYSAAFR [M+2H]−98 400 600 800 1000 1200 1400 1600 100 200 300 400 m/z Intensity

• b_0(15)2+

b*(14)2+ b*(6)1+ b_0−98(8)1+ 773.07 716.26 646.04 761.24 y*−98(8)1+ y(6)1+ y−98(8)1+ y*−98(17)2+ 735.47 678.3 752.74 755.18 b_0(19)2+ b−98(9)1+ b*(20)2+ b*−98(17)2+ 916.09 906.78 973.1 803.54 y(8)1+ y(9)1+ y*−98(10)1+ y(17)2+ 850.5 963.58 920.05 811.84 b*(23)2+ b(11)1+ b(21)2+ b_0(21)2+ 1108.29 1189.54 1039.29 1030.1 y(12)1+ y(11)1+ z+1(4)2+ y(22)2+ 1148.54 1091.45 1211.44 1075.6 b−98(13)1+ b(12)1+ b(27)2+ b(13)1+ 1249.77 1290.54 1318.63 1347.69 y(14)1+ y_0(13)1+ y_0(27)2+ 1363.76 1231.51 1331.75 b−98(15)1+ 1464.93 y(16)1+ y*(16)1+ 1521.63 1504.7 title: peakplot query number: q12787_p1 mascot Score: 119.88 precursormass/charge: 937.786450,3+ delta score; pep rank1 − pep rank2: 95.53 peptide sequence: MKDLSSNTQALTGTNTGGAIDGVSTTDR [M+3H]−98 400 600 800 1000 1200 1400 50 100 150 200 250 m/z Intensity

• b_0−98(14)2+

b_0(11)2+ b_0−98(9)2+ b_0−98(11)2+ 514.47 434.2 312.29 384.12 y(5)1+ y*(5)1+ y_0(6)2+ y(4)1+ 433.42 416.53 289.48 376.29 b(8)1+ b_0(9)1+ b_0(8)1+ b(9)1+ 653.25 722.34 635.36 740.3 y(7)1+ y_0(13)2+ z+1(5)2+ y(6)1+ 683.38 617.3 704.23 596.34 b_0(20)2+ c(10)1+ b_0(10)1+ b_0(12)1+ 853.08 844.1 809.33 953.97 y(10)1+ y(8)1+ y(9)1+ y(18)2+ 914.51 770.44 857.58 798.95 b_0(13)1+ b_0(14)1+ b(13)1+ 1040.42 1127.09 1058.43 y(12)1+ y(11)1+ y_0(12)1+ y*(12)1+ 1088.47 1001.48 1070.52 1072 b(15)1+ b_0(17)1+ b_0(15)1+ 1308.44 1434.49 1290.31 y(13)1+ y(15)1+ y(16)1+ y*(15)1+ 1251.57 1365.57 1422.52 1348.1 title: peakplot query number: q8036_p1 mascot Score: 119.51 precursormass/charge: 870.856500,2+ delta score; pep rank1 − pep rank2: 114.15 peptide sequence: GSSSGGGYSSGSSSYGSGGR [M+2H]−98

SLIDE 39

500 1000 1500 100 200 300 400 500 600

m/z Intensity

• a(7)2+

b*(5)2+ c(6)2+ b_0(6)2+

363.24 284.09 357.43 339.24

y−98(8)2+ y_0−98(10)2+ y*(6)2+ y_0−98(8)2+

419.49 488.5 367.52 410.92

a*(7)1+ b*(6)1+ c(12)2+ b−98(7)1+

708.73 679.44 649.49 655.78

z+1(9)2+ y_0(12)2+ y(5)1+ y*−98(5)1+

695.58 629.97 665.01 550.13

b(7)1+ b*−98(8)1+ a*(15)2+ b*(17)2+

754.16 793.94 760.27 845.98

y(6)1+ z+2(16)1+ y_0(8)1+ y*−98(18)2+

750.77 737.03 919.18 929.6

b(9)1+ b(19)2+ b_0−98(20)2+ a_0(21)2+

1037.45 997.51 1012.05 1125.32

y_0(9)1+ z+1(4)2+ y−98(11)1+ y_0−98(19)2+

1016.25 979.2 1093.33 985.14

b*(11)1+ a*(12)1+

1206.64 1235.72

y(11)1+

1190.2

title: peakplot query number: q10882_p1 mascot Score: 0.02 precursormass/charge: peptide sequence: DIIKLLGRQSVGPDASGWVFR

Intensity

SLIDE 40

summary

contribution

◮ peakplot enables large scale high throughput labeling of

tandem mass spectra

◮ easy to adapt and to plugin into existing software, e.g., LIMS ◮ open source code ◮ no commercial libraries nessesary

availability

◮ source:

svn co https://peakplot.svn.sourceforge.net/svnroot/peakplot peakplot

◮ CGI: http://fgcz-peakplot.uzh.ch ◮ LIMS: http://fgcz-bfabric.uzh.ch

SLIDE 41

thanks

special thanks to:

◮

Ralph Schlapbach (general support)

◮

Bertran Gerrits (peakplot)

◮

Jonas Grossmann (discussion and slides)