protein/peptide characterization
DESCRIPTION
Protein/Peptide characterization. Peptide Mass Fingerprinting. HUMAN Carbonic anhydrase I. - PowerPoint PPT PresentationTRANSCRIPT
Protein/Peptide characterizationPeptide Mass Fingerprinting
mass position peptide sequence
4541.241 174-213APFTNFDPSTLLPSSLDFWT YPGSLTHPPLYESVTWIICK
2796.291 90-113 LFQFHFHWGSTNEHGSEHTV DGVK
2759.389 228-252 SLLSNVEGDNAVPMQHNNRP TQPLK
2256.042 58-76 EIINVGHSFHVNFEDNDNR
1929.007 40-57 HDTSLKPISVSYNPATAK
1742.907 19-34 LYPIANGNNQSPVDIK
1612.786 114-127 YSAELHVAHWNSAK
1580.791 214-227 ESISVSSEQLAQFR
1186.686 138-149 ADGLAVIGVLMK
1153.479 1-10 ASPDWGYDDK
1026.510 128-137 YSSLAEAASK
985.437 81-89 GGPFSDSYR
970.593 160-168 VLDALQAIK
945.442 11-18 NGPEQWSK
714.378 150-156 VGEANPK
565.282 35-39 TSETK
HUMAN Carbonic anhydrase I
MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF
Ion Sources
Matrix-assisted Laser Desorption/Ionization (MALDI)
Mass Analyzers
“MS-only”
Time-of-Flight (Tof)
MALDI plate+ 20,000 V
Nitrogen Laser337 nm
Detector-20,000 V
d
Field-free drift region
+++++
+ ++ ++++ ++
Mass AnalyzersReflectron Tof, orthogonal injection
Reflectron Tof – increase in resolution
Delayed Extraction – increase in resolution
Inte
nsi
ty
Mass to Charge ratio (m/z)432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.8
0
1000
2000
3000
4000
5000
6000
7000
8000
432.9226
433.2556
433.5887
m/z = 0.33
m/z = 0.33
Angiotensin: DRVYIHPFHL C62H89N17O14
“Monoisotopic Peak”12C62
1H8914N17
16O14
X + 1 peak12C61
13C11H89
14N1716O14
+12C62
1H8914N16
15N116O14
X + 2 peak12C60
13C21H89
14N1716O14
+12C61
13C11H89
14N1615N1
16O14
+12C62
1H8914N16
16O1318O1
m = 1 Da, z=3
400 500 600 700 800 900 1000 1100 1200 1300m/z, amu
0
8000
Inte
nsity, coun
ts
432.9226
Type Element Symbol Integer Mass1
Exact Mass2
Abundance X+1 Factor3
X+2 Factor4
X Hydrogen H 1 1.0078 99.99
D or 2H 2 2.0141 0.01
X+1 Carbon 12C 12 12.0000 98.91
13C 13 13.0034 1.1 1.1nC 0.0060nC2
X+1 Nitrogen 14N 14 14.0031 99.6
15N 15 15.0001 0.4 0.37nN
X+2 Oxygen 16O 16 15.9949 99.76
17O 17 16.9991 0.04 0.04nO
18O 18 17.9992 0.20 0.20nO
X Phosphorus
P 31 30.9738 100
X+2 Sulfur 32S 32 31.9721 95.02
33S 33 32.9715 0.76 0.8nS
34S 34 33.9679 4.22 4.4nS
X+2 Chlorine 35Cl 35 34.9689 75.77
37Cl 37 36.9659 24.23 32.5nCl
DRVYIHPFHL C62H89N17O14
[M+3H]3+ = 432.9226 x 3 = 1298.768Measurement mass error = ~50 ppm
C62H89N17O14
X + 1 peak12C61
13C11H89
14N1716O14
+12C62
1H8914N16
15N116O14
+12C62
1H882H1
14N1716O14
68%
6.3% } 75%
432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.80
1000
2000
3000
4000
5000
6000
7000
8000
77%
0.9%
Electron = 0.000549 g/molProton = 1.007253 g/molNeutron = 1.00864 g/mol
Protein/Peptide characterizationPeptide Mass Fingerprinting
mass position peptide sequence
4541.241 174-213APFTNFDPSTLLPSSLDFWT YPGSLTHPPLYESVTWIICK
2796.291 90-113 LFQFHFHWGSTNEHGSEHTV DGVK
2759.389 228-252 SLLSNVEGDNAVPMQHNNRP TQPLK
2256.042 58-76 EIINVGHSFHVNFEDNDNR
1929.007 40-57 HDTSLKPISVSYNPATAK
1742.907 19-34 LYPIANGNNQSPVDIK
1612.786 114-127 YSAELHVAHWNSAK
1580.791 214-227 ESISVSSEQLAQFR
1186.686 138-149 ADGLAVIGVLMK
1153.479 1-10 ASPDWGYDDK
1026.510 128-137 YSSLAEAASK
985.437 81-89 GGPFSDSYR
970.593 160-168 VLDALQAIK
945.442 11-18 NGPEQWSK
714.378 150-156 VGEANPK
565.282 35-39 TSETK
HUMAN Carbonic anhydrase I
MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAKEIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSAELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEGDNAVPMQHNNRPTQPLKGRTVRASF
Electrospray Ionization (ESI)
550 600 650 700 750 800 850 900 950 1000 1050m/z, amu
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
Intensity, counts
719.6340
539.9927
563.9729
684.9795
1079.4690597.6693670.5904
540.0 540.4 540.8 541.2m/z, amu
Inte
nsity, coun
ts
539.9927
540.2492
540.4936
540.7446
540.9927
1079.0 1080.0 1081.0m/z, amu
Inte
nsity, coun
ts
1079.46901078.9731
1079.9691
[M+2H]2+
[M+3H]3+
[M+4H]4+
Protein/Peptide characterization
Collision-activated dissociation (CAD)
N
R1
O
N
R2
O
H
N
R1
O
N
R2
OH H
+
R1
O
+ + N
R2
OH2
“b” “y”
H
H
N
H
R1
O+N
H
R1+N
H
“a”
+TOF Product (432.9): Experiment 2, 15.915 min from fmw_angiotst_030105_2.wiffa=3.55571269460828870e-004, t0=1.03585549568179790e+001
Max. 1654.0 counts.
150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z, amu
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
Inte
ns
ity, c
ou
nts
647.3574
392.7211
382.1879269.1692
378.7201619.3550
513.2865257.1463
132.1045 325.6743 371.2152 500.7836 784.4101569.3014217.1281
D R V Y I H P F H Lb-type 116 272 371 534 647 784 881 1028 1165 1296
y-type 1296 1181 1025 926 763 650 513 416 269 132
y2
y4
y52+y1
b4
b5
b6
b62+
a5a8
2+
y62+
a62+
Introduction to Mass Spectrometry
Amino Acid Residue Masses
Amino Acid3 Letter
CodeSingle Letter
Code
Residue Mass
Monoisotopic Average
Glycine Gly G 57.02147 57.052
Alanine Ala A 71.03712 71.079
Serine Ser S 87.03203 87.078
Proline Pro P 97.05277 97.117
Valine Val V 99.06842 99.133
Threonine Thr T 101.04768 101.105
Cysteine Cys C 103.00919 103.144
Isoleucine Ile I 113.08407 113.160
Leucine Leu L 113.08407 113.160
Asparagine Asn N 114.04293 114.104
Aspartic Acid Asp D 115.02695 115.089
Glutamine Gln Q 128.05858 128.131
Lysine Lys K 128.09497 128.174
Glutamic Acid Glu E 129.04260 129.116
Methionine Met M 131.04049 131.198
Histidine His H 137.05891 137.142
Phenylalanine Phe F 147.06842 147.177
Arginine Arg R 156.10112 156.188
Tyrosine Tyr Y 163.06333 163.170
Tryptophan Try W 186.07932 186.213
+TOF Product (578.3): Experiment 5, 66.365 min from 03250_1433beta_12hr.wiffa=3.55560354546847490e-004, t0=1.01409857317689070e+001
Max. 5229.0 counts.
160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
Inte
ns
ity, c
ou
nts
329.1613
255.1456175.1256
272.1750
312.1307
157.1041
158.0911 270.1291257.1671 330.1647 442.2415344.1971212.1467
245.1295217.1389183.1487 354.2145 425.2125313.1268 470.3016383.1911195.1234
+TOF Product (578.3): Experiment 5, 66.365 min from 03250_1433beta_12hr.wiffa=3.55560354546847490e-004, t0=1.01409857317689070e+001
Max. 5229.0 counts.
500 550 600 650 700 750 800 850 900 950 1000m/z, amu
50
100
150
200
250
300
350
400
450
500
550
600
650
Inte
ns
ity, c
ou
nts
714.4382
686.3713570.8088
785.4351757.4333827.5163
658.3663
740.3993 856.4853641.3310548.8108 617.3710 669.3386884.4812 1000.5370768.3821
R G D L P F V V P R157 214 329 442 539 686 785 884 981 1155
1155 999 942 827 714 617 470 371 272 175 y
b
m/z300 600 900 1200 1500
12
3
45
MS Scan
200 400 600 800 1000 1200m/z
Similar to LIM and SH3 protein 1
MS/MS #1
400 800 1200 16001800m/z
???
MS/MS #2
200 400 600 800 1000 1200m/z
Acyl-CoA-binding protein
MS/MS #3
200 400 600 800 1000 1200m/z
hypothetical protein MGC2477
MS/MS #4
200 400 600 800 1000 1200m/z
skeletal muscle tropomyosin
MS/MS #5
MS scan
MS/MS#1 MS/MS#2 MS/MS#3 MS/MS#4 MS/MS#5
~10 seconds
Mass AnalyzersQuadrupole Mass Spectrometer
+
+--
0 = U – V cos t
Ion source
lens
lens
quadrupole detector
U = fixed (DC) potentialV cos t = applied RF, amplitude V and frequency t
Mass AnalyzersTriple Quadrupole
“Single quad”
“Triple quad”
Ion source
lens
lens
quadrupole detector
lens
quadrupole
lens
quadrupole
Q1 Q2 Q3
Why triple quadrupole? - MS/MS analysis of peptides
lens quadrupole detectorquadrupole quadrupole
Q1 Q2 Q3
Pass 1 m/z valueFragment, pass all fragment ions
scan m/z values (single quad)
Mass Analyzers
Quadrupole Ion Trap
Thermo-Finnigan LTQ mass spectrometer
Quadrupole Time-of-Flight
ITMS + c ESI Full ms
400 500 600 700 800 900 1000 1100 1200
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
816.63
432.30
783.95
778.23701.98
593.28
743.58 857.95 1223.77
526.87 1166.32887.06620.07453.33 1114.11954.77 1024.82
1140 1145 1150 1155 1160 1165 1170 1175 1180
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1166.32
1174.80
1167.211143.44
1175.88
1168.221144.31
1176.781173.91
1153.831165.691148.21 1156.90 1182.83
LTQ Ion trap
+2? +3?
+4
+3+5
0.87
420440460480500520540560580600620640660680700720740760780800820840860880m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
997
Inte
nsity, coun
ts
496.8928
445.2570
543.7190451.2044
539.5885
487.5576
744.8367
493.0928
601.6043 730.8341591.2612
487.0488.0489.0490.0491.0492.0493.0494.0495.0496.0497.0498.0499.0500.0501.0502.0m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
997
Inte
nsity, coun
ts
496.8928
497.2254
487.5576
497.5597487.8905
493.0928
488.2278497.8955
+3
+3
+1
0.33
0.33
Quadrupole Tof
Fourier Transform Ion Cyclotron Resonance Mass Spectrometer(FTMS)
resolution = 5,000,000
Introduction to Mass Spectrometry
432.4 432.6 432.8 433.0 433.2 433.4 433.6 433.8 434.0 434.2 434.4 434.6 434.80
1000
2000
3000
4000
5000
6000
7000
8000
Dynamic Range
400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, amu
0500
1000150020002500300035004000450050005500600065007000750080008490
Intensity, counts
432.9226
382.2313 463.7657648.8623
513.2979 858.5471
636.0 637.0m/z, amu
0
49
Intensity, counts
635.7516
636.2614
resolution = 11,000
Resolution
resolution = 5,000,000
m/mm = FWHM