04_Altelaar 2014 - Quantification_1

Quan%ta%ve proteomics Maarten Altelaar, 2014 Proteomics Altelaar et al. Nat Rev Gen 14, 2013, 35-‐48 Quan%ta%ve proteomics Quan%ta%ve proteomics Control Diseased, s%mulated, Knock down, etc. How quan%ta%ve is mass spectrometry? Quan1ﬁca1on requires internal standards or controlled condi1ons • All pep%des and proteins will experience DIFFERENT levels of loss during a proteomic experiment • In most cases the level of sample loss changes in each proteomic experiment • Stable Isotope labelling was introduced to overcome the above obstacles. • Isotopomeric pep%des behave almost iden%cal throughout the experiment EXCEPT for the mass spectrometric analysis. Basics : Natural Elemental Isotopes In nature, most elements are present in more than one isotopic form: e.g. Carbon is present in two stable forms 12C and 13C and one unstable 14C which is radioac%ve and used for ‘carbon da%ng’. Mass spectrometry can dis%nguish these isotopes since they have diﬀerent mass Isotope
Relative
Abundance
Isotope
Relative
Abundance
Carbon
12C
100
13C
1.11
Hydrogen
1H
100
2H
.016
Nitrogen
14N
100
15N
.38
Oxygen
16O
100
17O
Sulfur
32S
100
33S
Chlorine
35Cl
Bromine
79Br
Element
Isotope
Relative
Abundance
.04
18O
.20
.78
34S
4.40
100
37Cl
32.5
100
81Br
98.0
Basics : Natural Elemental Isotopes Appearance of spectra: C20H40 CH4 CH4: C1 H4 p(gss, s/p:40) Chrg 1R: 1000 Res.Pwr. @FWHM
100
CH3Cl C20H42: C20 H42 p(gss, s/p:40) Chrg 1R: 10000 Res....
16.03
100
CClH3: C1 Cl1 H3 p(gss, s/p:40) Chrg 1R: 10000 Res...
282.33
100
95
95
90
90
85
85
80
80
75
75
70
70
65
65
60
60
49.99
13CH 35Cl 3
95
90
85
80
70
65
55
50
45
60
55
50
45
40
40
35
35
13CH
30
25
Relative Abundance
Relative Abundance
Relative Abundance
75
12C
30
4 15
15
10
10
5
5
17.03
18.04
0
16.0
16.5
17.0
m/z
17.5
18.0
45
35
13CH
30
25
283.33
20
50
40
13
19 CH40 25
20
13CH 37Cl 3
55
51.99
3Cl 20
12C
13
18 C2H40 15
10
5
284.33
51.00
0
282.5
283.0
283.5
m/z
284.0
284.5
285.0
50.0
50.5
51.0
52.99
52.00
0
51.5
52.0
m/z
52.5
53.0
54.00
53.5
54.0
ADTQLLLLR Isotopically labeled Ra%o = 2 Unlabeled Mass diﬀerence Where and How to Introduce Stable Isotopes Biological incorpora1on Chemical incorpora1on (SILAC/Metabolic) (ICAT)
(iTRAQ, MassTag) Absolute quan%ﬁca%on instead of rela%ve quan%ﬁca%on can be performed if the level of the internal standard is known. SILAC:Stable Isotope Labeled Amino Acids in Culture • Incorpora%on of ‘heavy’ isotopes is performed by uptake of amino acids by the cells from the media • Require the cell type to be auxotrophic for that amino acid. • Typical amino acid(s) chosen for SILAC are: • Leucine; one of the most common amino acids present in pep%des • Lysine and Arginine; all pep%des will have at least one labeled amino acid if trypsin is used.
e.g. 12
13
C
C
13
C
C 13C
12
12
C
13
C
O
H2N 13C
OH
13C
12
6 leucine C12
C
12
H2N
C12 O
C
OH
regular leucine SILAC:Stable Isotope Labeled Amino Acids in Culture SILAC:Stable Isotope Labeled Amino Acids in Culture Light Heavy Certain cell types can convert arginine into proline. In those systems pep%des that contain prolines will have mul%ple peaks for the ‘heavy’ pep%de. + Log ra%o reversed Label swap unchanged -‐ + Log ra%o forward -‐ Speciﬁc A B B A Pijnappel et al. Nature 2013 495(7442):516-‐519. A
B
Metabolic Labeling of Arabidopsis thaliana 1: Metabolic labeling with 15N (KNO3, <5% NH3) 14N 14N 15N • Requires cell types that do not extract amino acids from the media • Typical media use: • 15N Potassium Nitrate for Arabidopsis thaliana • 15N Ammonium Sulfate for Sacchromyces Cerevisiae Metabolic Labeling of C. elegans Metabolic Labeling of Mammals SILAC Labeling of Mammals Quan1ta1on through Chemical Deriva1sa1on There are many, many,...many ways and all have advantages and disadvantages. ICAT Chemical labeling : the iodoacetyl group reacts selec%vely with Cys residues of proteins. ICAT Reagents: Heavy reagent: d8-‐ICAT (X=deuterium) Light reagent: d0-‐ICAT (X=hydrogen) O N N S Bio1n tag O X X N O X X O O X X X X Linker (heavy or light) O N I Thiol speciﬁc reac1ve group Reagent has been modiﬁed to use 13C instead of 2H or D due to deuterium containing pep%des elu%ng earlier in reverse phase chromatography than their ’normal’ analogues. Dimethyla1on formaldehyde peptide
+
NH2
X
X
peptide
O
N
+
H
X
X
O
H
NaCNBH3
peptide
H
O
H
+
N
X
X
X
X
+
H
peptide
N
H
X
H
+
X
O
X
X
NaCNBH3
X
peptide
N
X
H
H
X
X
• Dimethylates lysine residues and N-‐termini. • Current experimental condi%ons: (100 mM TEAB or sodium acetate) • Semi-‐tolerant towards ‘dirty samples’. (Some types of sample can ini%ate polymerisa%on.) • Allows labelling of whole digests in 10mins. Dimethyla1on RT: 9.21 - 52.05
90
NL: 3.68E6
Base Peak F:
ITMS + c NSI Full
ms
[350.00-1500.00]
MS
060825_SM3017_
14
24.49
Normal 100
Relative Abundance
80
70
27.33
60
28.90
34.02
33.22
37.55
37.96
50
29.66
40
30
35.54
40.75
31.81
39.19
25.01
42.15
20
44.06
10
11.39
0
100
15.03
18.89
46.92 47.48
22.21 23.85
24.97
NL: 1.91E6
Base Peak F:
ITMS + c NSI Full
ms
[350.00-1500.00]
MS
060825_sm3017_
15
38.28
90
Dimethyla%on 80
35.15
32.88
70
28.62
60
50
24.63 25.22
40
35.60
31.71
31.24
41.13
26.92
30
37.39
40.32
41.80
43.15
20
10
0
50.74
29.30
10.07 12.33
10
14.96 16.27
15
19.87 21.81
20
44.75
24.49
25
30
Time (min)
35
Likle change in hydrophobicity of methylated pep%des. Not necessary to clean up sample aler labelling. Likle change in MSMS spectra. 40
45
49.22 49.45
46.59
50
On-‐line dimethyla1on SoZware 678.34
z=2
100
Peak picking Peak integra%on Mass accuracy Resolu%on 80
Relative Abundance
• 
• 
• 
• 
90
680.35
z=2
678.84
z=2
70
60
681.80
z=2
676.33
z=?
50
40
682.30
z=2
676.83
z=?
30
20
0
674
682.81
z=2
677.33
z=?
675.47
z=?
10
679.34
z=2
676
685.83
z=2
686.33
z=2
686.83
z=2
684.21 685.26
z=?
z=?
678
680
682
684
686
m/z
623.79
z=2
100
90
RT: 42.86 - 125.78
53.82
90
Relative Abundance
53.65
100
64.05
80
74.19
74.14
60
50
30
20
86.38
47.70
10
632.86
z=2
40
30
628.31
z=2
622.37
z=?
616.28 618.33
z=?
z=?
615
98.93
101.25
637.86
z=?
636.80
z=?
639.33
z=2
643.05
z=1
67.87
620
625
630
m/z
635
640
645
122.34
108.77
59.17
50
0
92.94
76.32
632.36
z=2
624.29
z=2
627.81
z=2
60
10
81.51
40
70
20
83.93
623.76
z=2
115.96
100
0
90
50
60
70
80
Time (min)
MS MSMS MS 90
100
110
120
Relative Abundance
Relative Abundance
70
638.32
z=2
80
80
624.27
z=2
70
60
50
40
624.77
z=2
30
20
10
0
620.28
z=?
622.37
z=?
620
622
625.27
z=2
624
626
627.79
z=2
629.76
z=?
628
630
m/z
631.81 632.84
z=2
z=?
632
634
635.97
z=3
636
639.31
z=?
638
640
OR4_110703_EDG_11EDEG007_IP_3d #7693 RT: 81.10 AV: 1 NL: 1.84E4
F: FTMS + p NSI Full ms [350.00-1500.00]
622.63
z=3
100
Relative Abundance
90
Relative Abundance
OR4_110703_EDG_11EDEG007_IP_3d # 7688 RT: 81.05 AV: 1 NL: 1.56E4
F: FTMS + p NSI Full ms [350.00-1500.00]
620.95
z=3
100
623.63
z=3
90
622.63
80
z=3
623.97
622.29
z=3
70
z=3
621.29
60
z=3
50
622.96
625.18
z=3
z=?
621.62
40
z=3
620.33
624.30
30
z=?
621.94
z=3
624.80
z=3
623.32
20
z=?
z=?
10
625.84
z=?
60
50
622.96
z=3
620.84
z=?
40
620.33
z=?
30
623.97
z=?
621.98
z=?
624.30
z=?
10
625.18
z=?
625.04
z=?
0
620
621
622
623
m/z
624
625
626
OR4_110703_EDG_11EDEG007_IP_3d # 7699 RT: 81.17 AV: 1 NL: 2.02E4
F: FTMS + p NSI Full ms [350.00-1500.00]
620.95
z=3
100
623.63 623.97
z=3
z=3
622.29
z=3
70
621.28
z=3
621.62
z=3
60
50
40
30
621.95
z=3
620.33
z=?
20
622.63
z=3
622.96
z=3
Relative Abundance
80
625.18
z=?
623.30
z=3
624.30
z=3
620
621
622
623
m/z
624
625
626
OR4_110703_EDG_11EDEG007_IP_3d #7703 RT: 81.21 AV: 1 NL: 1.49E4
F: FTMS + p NSI Full ms [350.00-1500.00]
622.63
z=3
100
622.29
621.28
z=3
z=?
90
623.63
80
z=3
90
Relative Abundance
623.63
z=?
621.62
621.28 z=3
z=3
70
20
0
624.97
z=?
70
620.95
z=?
60
621.62
z=?
622.96
z=3
50
620.33
z=?
40
30
623.97
z=3
623.30
z=3
621.82
z=?
20
10
625.18
z=?
624.30
z=3
625.67
z=?
10
0
0
620
621
622
623
m/z
624
625
626
OR4_110703_EDG_11EDEG007_IP_3d # 7709 RT: 81.27 AV: 1 NL: 1.50E4
F: FTMS + p NSI Full ms [350.00-1500.00]
621.28
622.63
623.63
620.95 z=3
100
z=3
z=3 623.97
z=3
z=3
90
622.29
80
z=3
Relative Abundance
622.29
z=3
80
70
624.30
z=3
60
50
621.95
z=3
621.62
z=3
40
30
620.83
z=?
20
623.30
z=3
622.96
z=3
625.18
z=?
625.81
z=?
10
0
620
621
622
623
m/z
624
625
626
620
621
622
623
m/z
624
625
626
Large scale quan1ta1ve proteomics analysis; stem cells Munoz et al. Molecular Systems Biology 2011 Tech Rep 2: ESC/iPS
Tech Rep 2: Progenitor/iPS
Dimethyl – Technical Reproducibility Tech Rep 1: Progenitor/iPS
Tech Rep 1: ESC/iPS

Download Report