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Anlise Protemica

Hugo Osrio
hosorio@ipatimup.pt

PROTEOMICS
Proteomics: Prote - Protein

Omics - Total

Protein identification
Characterization: Post-translational modifications PTMs

Papers/year (Proteomics, PubMed)

Cells

2011 5428
2001 522
1998 22
1997 1!

Organs
Organisms

Tissues

Cancer

Medical research

Neurosciences

Psychiatry

Cardiology

Biological research

Yeast

Plants

Fish

IPATIMUP Proteomics Unit

2D gel electrophoresis
Separation of Human Serum proteins by 2D gel electrophoresis
Isoelectric point
3
MW (kDa)
250
150
100
75

50

37

25

10

2D Gel Analysis: Progenesis Samespots

www.nonlinear.com/products/progenesis/samespots/overview/
www.nonlinear.com/products/progenesis/samespots/downloads/

2D Gel Analysis: Progenesis Samespots


Quality control

2D Gel Analysis: Progenesis Samespots

Alignment

2D Gel Analysis: Progenesis Samespots

Results

2D Gel Analysis: Progenesis Samespots

Data analysis

2D Gel Analysis: Other Softwares

www.biorad.com/prd/en/US/LSR/PDP/966de
b78-2656-437f-b7a4ab0a9bd45c8d/PDQuest-2-DAnalysis-Software

www.gelifesciences.com/webapp/
wcs/stores/servlet/productById/en/
GELifeSciences-PT/28940830

Mass spectrometry

Inside a Mass spectrometer

Ionization Source

Mass analyzer

Generates ions

Separates ions

Detector
Detects ions and
amplifies the signal

MALDI Matrix Assisted Laser Desorption Ionization


ESI ElectroSpray Ionization

TOF Time of Flight


Orbitrap
Quadrupole
Fourier transform ion cyclotron resonance FT-ICR

Sample + Matrix

+
+

MALDI
Matrix Assisted Laser Desorption Ionization

+
+

-Cyano-4-hydroxycinnamic acid
Sinapinic acid
2,5-dihydroxybenzoic acid

Matrix Assisted Laser Desorption Ionization

MALDI

Laser

Voltage (20 kV)

Time Of Flight (TOF)

mass/charge

Detector

Koichi Tanaka MALDI Ionization


John Fenn ElectroSpray Ionization

Molecular Weight of entire proteins


Apomyoglobin

4700 Linear Spec #1[BP = 16956.1, 1251]

16953.3 Da

100

1 2 5 1 .1

90

80

70

% In te n s ity

60

50

40

30

Thioredoxin
11674.5 Da

20

10

0
9 9 8 5 .0

1 1 6 3 1 .4

1 3 2 7 7 .8

1 4 9 2 4 .2
M a s s (m /z )

1 6 5 7 0 .6

1 8 2 1 7 .0

Molecular Weight of peptides


MS - INTERNAL CALIBRATION

Plate: 1 Spot: F2

120000

1821.91

1821 Da

110000

100000

1688 Da
90000

1688.86

948 Da
948.45

80000

1781.86

C l u s te r A r e a

70000

1733.86

60000

50000

2132.06

1659.84

40000

1958.06

30000
1388.77

20000

3836.78
764.43
893.45

10000

1025.51

1073.52

1611.85

823.48
1641.76

842.51
871.48

1028.94
1089.51
970.44 1116.52

1578.83

1402.78

1825.90

2211.11

1888.93
1919.96

1488.70

3850.84

2445.01

2225.12

2807.32

2189.09

1878.93

3014.48

0
1000

1500

2000

2500
Mass

3000

3500

Molecular Weight of glycans

Molecular Weight of Polymers

4700 Linear S pec #1=>AdvBC(32,0.5,0.1)[BP = 5541.6, 5282]


5541.1548

100

5281.6
5673.1226

5761.1216

90
5409.1899
5365.4541

5805.1416
5892.9102

80

5849.4219
5321.2124
5277.4556

70

5937.0415
5980.6997
6024.8804

% Intensity

60
5189.7905
5145.6392

6068.6787
6113.2207

50

6157.3066

5101.9717

40

6245.3008

5014.1318
4970.2163

6288.8379

30

20

10

0
2991

4018

5045

6072
M ass (m/z )

7099

8126

Protein identification
2D Gel
+

pI

MW

Excision of Spots of interest


Spot Excision

Membrane transfer

Western Blot

Antibodies to specific proteins /


Post Translational Modifications

Protein Identification by a gel-based approach

Peptide extraction

Peptide mass spectra


Final - Shots 625 - training january; Run #1; Label A4
928.371

100

7.3E+4

90

80

929.370
70

60

50

40

930.371
30

944.362

20

910.366

Protein

10

0
860

931.371

945.368
912.361
932.368
946.371
960.359
898.471
882.529
938.756
914.369
920.867
868.501
927.758
953.767 967.531
905.508
875.680
891.042
892

924

956

Mass (m/z)

Protease in gel digestion


700 Da
200 Da

Bioinformatic analysis
1050 Da

900 Da

Trypsin: Cuts at lysine and arginine


Lys_C: Cuts at lysine
PepsinA: Cuts at phenylalanine and leucine

996.546
982.551
989.823
988

1009.215
1020

Peptide Mass FingerPrint

Experimental mass spectrum


Final - Shots 5040 - 11Oct 2010; R un #30; Label E3
100

1298.651( R 11281,S906)
1.5E+4

90

80

in silico enzimatic digestion


Theoretical mass spectra

70

60

50

1088.519( R 9327,S508)
1210.607( R 10791,S460)
1901.838( R 17349,S332)
880.417( R 8953,S490)

40

1906.829( R 16997,S261)
898.442( R 7544,S370)

MS - INTERNAL CALIBRATION
340000

1994.885( R 18017,S233)
2126.984( R 17889,S218)

30

Statistical analysis

Plate: 1 Spot: F9

320000
1213.64

300000

1377.615( R 11118,S185)

280000
1835.96

260000

1771.929( R 16092,S138)

20

240000

1136.615( R 9246,S155)
220000

1361.8

2024.6

2687.4

3350.2

Plate: 1 Spot: E3

200000

C l u s te r A r e a

220000

180000

MS - INTERNAL CALIBRATION

Plate: 1 Spot: H1

160000

200000

1298.65

140000

200000

842.51

180000

896.47

120000

4013.0

100000
160000

Mass (m/z )

80000

180000

1540.73

1287.63

60000

140000
2211.10

40000
842.51

1153.61

1243.65

20000
926.48

1082.58 1181.61

788.41

C l u s te r A r e a

719.35

818.41

1034.54

3168.50

1882.02

940.49

120000

1117.63

1375.62

1475.77

2225.12

1818.93

1592.85

1338.70 1451.74
1307.69
1666.82
1269.68 1389.65 1564.79
1624.87

1791.75
1896.03
1761.87 1935.98
1983.02

2383.95 2490.19
2593.12
2337.14
2457.16
2275.01
2316.13

160000

2705.15
3223.32

0
1000

1500

2000

100000

MS - INTERNAL CALIBRATION

2500

3000

3500

Mass
Plate: 1 Spot: H5

160000

80000
150000

140000

842.51

140000

870.54

60000

927.49

1062.55

120000

120000

40000

1479.77
745.33

1011.49

1286.65

110000

1639.90

830.46

1880.90

1234.66

MS - INTERNAL CALIBRATION

1274.60

896.47

20000

1003.56

763.39

1320.57

1161.59
1995.93
1045.55

797.42

100000

1088.52

962.49

C l u s te r A r e a

1920.91

1592.81

1521.75

1439.74

90000

Plate: 1 Spot: H3

1567.72
1419.65

1390.66

1126.50

927.49

1439.75

1213.61
1193.59 1357.68

1062.52

280000

1538.77

2211.11

1707.75
2225.12
1858.87
2343.21
2096.10 2125.94
2496.38
1677.77 1793.96 1982.93
2282.09
1954.02 2062.92
2383.97
1747.79 1716.80 1816.90
2161.00 2258.01
2033.92
2420.09

2612.21
2545.17
2572.37

2801.34
2748.40
2718.17

1088.52

3083.49

3246.62

1900.84

1210.61

100000
2874.46

3408.67

1639.93

260000

C l u s te r A r e a

1904.93

130000

1000

1500

2000

2500

3000

3500

1427.68

4000

Mass

80000

828.33

240000
70000

870.55

220000

80000

60000

880.42

927.50

200000

1993.88

50000

2062.94

1612.89

40000

180000

C l u s te r A r e a

60000

1319.68
1287.67

30000

160000

1003.59

2125.93

719.37

842.51
805.40

2328.07

1904.95

1083.58

1770.93

1479.80
1516.64

960.56

1298.65

875.51

982.46

20000

1149.59

140000

1563.86

1915.95

1193.59
1140.59
1029.57

120000

1251.66
1211.59

1213.63

MS - INTERNAL CALIBRATION

1677.80

1355.69
1399.70

1660.71

1793.88
1719.87

Plate: 1 Spot: H18

1951.96

2062.94

2495.26
2196.11

2395.13

2046.00

1835.93

2153.94

1735.83 1771.92

1000

1003.59

2125.98
1612.78

2874.38
2011.93

1428.69

100000

1253.62

1523.81

1114.63

898.45

2096.10

1880.93

759.32

10000

1374.50

2211.11

1639.94

1088.52

2282.05

2511.02

2612.19

40000

3194.33

2541.28
2574.38

2786.68
2749.36

3045.47

3350.43
3095.34

1136.61

3280.43

1505.72

1500

2000

2500

896.47

3000

842.51

2496.34

3500

Mass

1456.68

65000
1287.66

80000

1179.60
830.45

20000
1612.78

1475.75

1062.54

60000

1029.58

1904.94

1791.73

1511.83

1320.60 1427.67
719.37
759.44

1835.95

1707.78

785.34

1677.79

2383.97

2496.34

50000

1377.61

20000

1552.67

1880.92
1819.89

1740.74
1561.75

1914.93

1657.81

45000

736.39

1393.60

2782.32
2211.11
2329.08
2705.19
2225.12
2289.99
2560.09
2082.99
2689.16
2399.05
2874.41
1952.96
2248.03 2343.00
2749.32
2096.08
2541.25
2817.36
2573.36

796.36
842.49

1475.76

1791.76

1657.80
1628.77

1522.72
1552.67

1707.78

1852.84
1975.93

1682.97

1861.84

3480.67

1000

3619.92

1500

2000

2500

3000

2084.00
2034.97

1734.80

1500

2290.00
2383.97
2212.12
2409.13
2157.98 2305.99
2271.03 2246.05 2335.07

2000

0
1000

1938.82

2689.16 2782.32
2549.08

3072.44

2717.15
2458.04

2569.17

2944.36
2799.29

3237.47
3312.34

2984.75

3312.38
3072.42 3174.52 3278.52
3223.35
3092.34

1179.60 1264.72 1357.47


992.46 1071.50
1331.55
1234.65
957.44
1107.56
863.41
921.48

2062.93
1993.88

1374.51

1124.57

1819.90

2125.97

785.33
1439.73

40000

1029.59

1274.60

55000

1916.83

1594.70

1226.58

60000

2500

3000

3500

Mass

3500

Mass
C l u s te r A r e a

40000

35000
1904.94

30000

MS - INTERNAL CALIBRATION

Plate: 1 Spot: H7
25000

170000

Final - Shots 5040 - 11Oct 2010; R un #30; Label E3


870.54

160000

842.51

20000

927.50

150000

100

745.30

15000

1479.78

1193.62
1107.55

10000

760.37

130000

962.53 1062.54
1003.59

786.30

896.46

1234.67
1161.62 1320.60

1493.74
1707.78
1346.64

1179.62

1996.91

1418.67

1747.78

1277.69

1043.54

90

1532.80
1387.71

1564.75
1612.81

1771.87
1678.84

1791.73

2139.00

2001.92

2334.98

3085.39

2545.17
2741.48

Plate: 1 Spot: H11

3035.25

3247.40
3246.52

3576.11

3959.72

0
1088.51

1000

1500

2000

2500

3000

3500

Mass

1003.58

100000
C l u s te r A r e a

2211.10

1858.91

1592.83

1298.63

110000
220000

1366.61

1.5E+4

1439.77

1082.59

5000

120000

MS - INTERNAL CALIBRATION

200000

1298.651( R 11281,S906)

1920.91

1639.92

816.45

140000

80

90000

1210.59

80000

180000
870.54
1179.58

70000

898.44

70

842.51

160000

60000

830.45

976.56

MS - INTERNAL CALIBRATION

Plate: 1 Spot: H10

50000

1475.73
927.49

140000

1029.57
1905.83

1287.63
1136.60

40000

280000

1791.71
1427.66

C l u s te r A r e a

1612.76
1213.63

120000

1088.52

1003.59

1377.59

797.38

30000

1993.86

2125.97

2383.97

1770.90
1914.85

778.44

2062.92

896.47

1060.55

20000

1275.64

770.36

240000

1337.67
1191.52

1639.91
1505.69

1707.76

2211.11

1835.94

1584.71

977.49

1657.77

1392.58

10000

2496.36

1900.82

1456.66

1261.55

1107.54

100000

1740.79

1676.71

1947.89
1985.92

2093.96

2400.05
2289.99
2226.13 2329.08

2541.20

2612.21

2478.06 2575.25

1000

1500

2000

2500

1901.838( R 17349,S332)

2123.04

870.54

880.417( R 8953,S490)

1179.61

927.49
719.36

180000

1195.57

898.44

1298.65

40

1458.70
1402.65 1493.74

1144.57
1034.59
1070.61

745.29

40000

160000

1003.58

842.51

1906.829( R 16997,S261)

1320.59
1707.78
1765.75
1586.79

1088.52

2281.04

1639.94

2652.20

1835.95

140000

1377.62
1118.55

1880.93

1434.76 1529.68

842.51
1559.77

120000

1684.88
1646.75

1136.61

1791.80
1811.95
1740.72

2383.96

1951.97

2211.11
1993.88

2717.10 2815.39

2343.00
2438.27
2264.59

2035.00
2060.94

880.42

2215.12

1918.85 2006.01

1003.58

65000

898.442( R 7544,S370)

1612.82

940.49
808.40

20000
70000

1088.519( R 9327,S508)
1210.607( R 10791,S460)

3500

1235.69

Plate: 1 Spot: H20

60000

75000

50

3223.43
3237.64

3000

Mass

200000
MS - INTERNAL CALIBRATION

1298.64

3073.51
2717.14

80000
1210.60

80000

3312.45

2782.36
2705.22

1856.84

1531.68

220000

85000

60

719.37

1298.65

260000

C l u s te r A r e a

0
699.0

1237.640( R 9786,S113)
2497.343( R 19048,S128)
1820.900( R 16302,S79)
1029.588( R 7897,S79)
1393.604( R 10852,S60)
2291.005( R 18508,S64)
1794.814( R 17757,S38)
921.478( R 8410,S55) 1412.212( R 6599,S20)
2690.148( R 17772,S47)
1997.895( R 14319,S29)
3074.423( R 15546,S48)
807.399( R 7419,S23)
1537.693( R 7392,S11)
2673.131( R 17546,S15)
1949.876( R 10399,S12)
3481.632( R 16563,S16)

2565.20

2461.19

2612.17
2579.22

1000

100000

1500

2000

2500

2975.43

3000

3132.57

1377.615( R 11118,S185)

3500

Mass
1029.58

1771.929( R 16092,S138)

1905.83
921.48

1493.73

1427.68

1140.54

1377.61
796.36

50000

1770.92

1237.64

60000

1179.59

1639.93

816.49

1639.94

1329.64

1657.79

1359.61
1537.77

1000

1500

785.34
1107.54

1993.87

1791.73

2125.97

3237.45

1456.68

3000

3500

1880.92

2496.32
2062.92

1521.68
1740.71
1684.84

1820.900( R 16302,S79)

2497.343( R 19048,S128)

1361.8

2024.6

2687.4

Mass (m/z )

1770.91
1594.68

1419.67

1985.98

2290.01

1815.88

2717.10

2282.04
2211.10

1854.81

3086.43

2248.02

1561.74

2542.21 2646.13

2084.00

2567.13

2780.27
2686.20

3073.36
3011.60

3312.30
3353.17

0
1500

2500

1029.588( R 7897,S79)
1393.604( R 10852,S60)
2291.005( R 18508,S64)
1794.814( R 17757,S38)
921.478( R 8410,S55) 1412.212( R 6599,S20)
2690.148( R 17772,S47)
1997.895( R 14319,S29)
3074.423( R 15546,S48)
807.399( R 7419,S23)
1537.693( R 7392,S11)
2673.131( R 17546,S15)
1949.876( R 10399,S12)
3481.632( R 16563,S16)

2383.96

1707.78

1357.71

1065.55

5000

1000

3072.47

2817.24

0
699.0

1905.83

20000

1254.61

2717.09
2782.30
2688.21

Mass

1427.68

980.50

2383.96
2211.10
2289.99
2078.91
2172.10 2282.08
1880.92
1923.99 2034.97
2247.95

1854.85

2000

1612.77
1029.58

761.41

1707.78

1584.72 1677.80

30000

10000

10

1819.89

1392.57

1071.50

830.45

1377.61

1791.73

1479.80

1264.70
982.45

20000
948.43

15000

1237.640( R 9786,S113)

1456.68

719.37
745.29

880.42

25000

1136.615( R 9246,S155)

2125.97

2062.93

1191.53

40000
1149.57 1320.59

40000

1234.67

2496.33

1505.71

1210.60
1088.52

898.44

1993.87

1612.77

745.32

45000

35000

20

80000

55000

1994.885( R 18017,S233)
2126.984( R 17889,S218)

30

2778.50
2681.31

2495.27
2364.23

2139.04 2297.03

927.49

60000

C l u s te r A r e a

10

MS - DEFAULT CALIBRATION

2000

2500
Mass

3000

3500

3350.2

4013.0

Bioinformatic analysis

Peptide Mass FingerPrint

Number of entries in UniProtKB

Swiss-Prot
(Reviewed)
Manual anotation

trEMBL
(Unreviewed)
Automatic anotation

Peptide sequencing by MS/MS


MS - INTERNAL CALIBRATION

Plate: 1 Spot: G8

MS spectrum

150000
842.51

1337 Da

140000

130000

1306.64

1156.59

120000

1337.69

110000

100000

1141.59

C l u s te r A r e a

90000

80000

70000
2211.11

60000

1268.69

999.53

1360.58

50000

1582.81

1430.75
787.51
810.53

2005.99
1280.69

846.49

40000

1058.52

1470.70

1201.63

742.47
1701.88

1373.67
1410.72

30000

1238.60

1045.56

1833.95

880.43

1635.80

896.43

1676.83

20000

1169.55
912.43

759.46

10000

2282.02

1791.86
1775.86

1453.68

1496.80
1110.54

1554.78

1007.46
950.48

1094.55

1212.57

1649.80 1728.85

2153.91

1874.02
1818.96
1940.91
1886.90

1613.72

1525.71

2390.21

2122.11

1744.84

973.49

1570.82

1920.95

2032.96
1947.99

2366.28

2225.11
2164.96

2039.98

2299.17
2192.13
2315.14
2255.15

1992.94 2072.02

2407.14

2349.26
2455.35

2587.33
2650.30

2802.66
2952.53
2796.43

0
800

1000

1200

1400

1600

1800

2000
Mass

2200

2400

2600

2800

3000

3200

Peptide sequencing by MS/MS

Timed ion selector


Detector

1337 Da
Collision cell
Intensity

m/z (Da)

Precursor peptide: 1337 Da

MS/MS - PRECURSOR 1337.6899 - DEFAULT CALIBRATION

Plate: 1 Spot: G8

70000

Peptide sequencing

F, a1

65000

60000

MS/MS spectrum

55000

50000
L

1337 Da

y1

45000

C l u s te r A r e a

1337.51

40000
a2

35000

30000

25000

20000

112.03

y4, VSTPL(b)

NQ(b)

15000

b2
PL(b)

NQV(b)

229.02
b3, TPLL(b)

TP(b), LL(a)

10000

442.08
171.03

496.18

155.02

y2 - NH3

5000
141.04

y4 - NH3

185.04

b4
839.34

YNQ(b)
y5, NQVSTP(a)

297.03

b5

1293.39
1329.75

967.41

y6

STPL(b)

914.30

0
100

200

300

400

500

600

700
Mass

800

900

1000

1100

1200

1300

MS/MS spectrum

b ions extend from the N-terminus


y ions extend from the C-terminus

Proteomics Bioinformatics Tools


Proteomics Resource Portal
Expasy

http://www.expasy.org/proteomics

Proteomics Bioinformatics Tools


Proteomics MS Research Tools
ProteinProspector

http://prospector.ucsf.edu/prospector/mshome.htm

Proteomics Bioinformatics Tools


Protein databases
UniProt

www.uniprot.org

Proteomics Bioinformatics Tools


Protein databases

NCBI

www.ncbi.nlm.nih.gov

Proteomics Bioinformatics Tools


Protein identification
Mascot

www.matrixscience.com

Proteomics Bioinformatics Tools


Theoretical protein digestion

PeptideMass

http://web.expasy.org/peptide_mass/

PeptideCutter

http://web.expasy.org/peptide_cutter/

Proteomics Bioinformatics Tools


MS/MS peptide fragmentation

Institute for Systems Biology MS/MS fragment ion calculator


http://db.systemsbiology.net/proteomicsToolkit/FragIonServlet.html

Proteomics Bioinformatics Tools


MS/MS peptide fragmentation
MS-Product, ProteinProspector
http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct

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