1000 Proteins Per Hour [pph] – Maximizing Protein ID From Complex Mixtures
Kai Scheffler1, Eugen Damoc2, and Thomas Moehring2
Thermo Fisher Scientific,
Scientific 1 Dreieich,
Dreieich Germany; 2 Bremen,
Bremen Germany
Results:
R
lt The
Th iincreased
d sensitivity
iti it and
d iimproved
d duty
d t cycle
l off the
th new
instrument enables a dramatic increase in the number of protein IDs with
increased sequence
q
coverage
g and high
g confidence.
B
A
EColi_noIIdet_30k_top10HCD_1#8704 RT: 55.52 AV: 1 NL: 6.11E5
T: FTMS+pNSIdFull [email protected][100.00-1420.00]
2% 30% ACN iin 60 min
2%-30%
i
Base Peak Chromatogram
RT: 17.51 - 87.64
36.76
100
30.04
22.13
80
36.67
39.68
33.83
22.23
60
33.74
29.99
36.80
41.51
34.67
41.58
29.95
22.00
21.13
21.02
40
20
51.01
51.07
50.96 51.15
55.90 56.06
50.91
55.83
56.14
51.30
41.45
39.51
24.18
28 11
28.11
42 37 45.15
42.37
45 15
24.27
53.46
45.31
42.57
NL:
3 66E8
3.66E8
Base Peak
MS
EColi_noIId
et 30k top2
et_30k_top2
0_CID_60mi
n_1
67.72
67.62
67.82
67.51
64.99
56.19 58.79
58.97
67.89
35
40
45
50
69.72
69.78
71.11
RT: 43.825 - 43.945
76.24
77.67
78.24
84.04
81 25
81.25
84.38
Sample Preparation
E. coli p
protein digests
g
were p
prepared
p
from a whole cell lysate
y
that was
reduced with DTT and alkylated
y
with iodoacetamide followed byy
digestion with a K/R
K/R-specific
specific protease for 16 h at 37 °C.
C.
65
70
75
80
85
1 OT Full MS scan + 20x dd CID scans
43.856
NL:
5.30E7
43.909
12
3.18 sec
Relative Abu
undance
8
Base Peak
MS
EColi_noIId
et_30k_top2
0_CID_60mi
n 1
n_1
43.921
43.832
43 834
43.834
43 873
43.873
43.868
43.836
43.843 43.847
43 84
43.84
43 85
43.85
43 86
43.86
43 87
43.87
43.900
43.894
43 88
43.88
43 89
43.89
43.943
43.927 43.931
43.898
43.890
43.885
43.851
0
43 83
43.83
43.881 43.883
43.941
43.935
43.907
43 90
43.90
43 91
43.91
43 92
43.92
43 93
43.93
43 94
43.94
Time (min)
EColi_noIIdet_30k_top20_CID_60min_1 #9130 RT: 43.86 AV: 1 NL: 5.28E7
T FTMS + p NSI F
T:
Full
ll ms [380
[380.00-2000.00]
00 2000 00]
562.9385
z=3
100
20
413.5692
z=3
712.9092
z=2
602.3181
z=2
557.7614
z=4
z
4
457.5734 482.7711
z=2
z
2
z=3
450
500
550
600
650
600
700
1009.5458 1124.5596
800
900
1000
1100
FIGURE 5.
5 Unique proteins (A) and unique peptides (B) identified
f
from
1 µg and
d 100 ng off E.
E coli
li digest
di
t run with
ith ddTOP20 and
d
ddTOP10 HCD methods (<1% FDR, 2%-30% ACN in 90 min).
1256.9731
1200
1300
1400
564.7686
643.4118
244.0922
329.1811
40
724.4352 825.4767 876.4540
972.5574
759.3694
992.0388 1132.0391
416.1776
20
200
300
400
500
600
700
800
m/z
900
1000
1100
A
1366.6221
1200
1300
1400
1500
843.9044
z=2
797.3939
z=2
888.1639
z=4
943.4906
z=2
1055.5076
z=2
2
700
750
800
850
900
1009.4714
1009
4714
z=4
950
1000
1050
1100
m/z
1400
729.0183
60
40
870.4299
618.6508
235.1070
332.1048
20
1200
744.8801
207.1122
567.7501
400
926.9
69765
600
800
1929.8
98708
1222.5409
1000
m/z
1200
1400
1600
1800
806
265.9941
40
234.1442
600
460.0355
279.0970
837.4692
623.3741
392.1806
213.1157
20
738.4023
701.3510 756.3984
200
300
400
500
600
700
919
946
.4628
800
400
1347.3
73011
900
1000
1100
1200
1300
EC
Cli
oli_noIIde
IId t_
t 30k
0kt
_top10HCD
D1#8
_1#8710 RT: 55
55
.54 AV
V1
: 1 NL
L: 2
212
.12E4
T: FTMS+pNSIdFull [email protected][100.00-1870.00]
677.3086
100
EColi_noIIdet_30k_top10HCD_1#8704 RT: 55.52 AV: 1 NL: 6.11E5
T: FTMS+pNSIdFull [email protected][100.00-1420.00]
Relative A
Abundance
e
Relative A
Abundance
e
Relative Abundance
A
m/z
80
60
40
221.0916 336.1184
394.1754 528.2807 629.3283
200
300
400
500
600
792.3927 893.4396 1008.4644
700
800
m/z
Relative A
Abundance
e
EColi_n
li noIIdet_
t 30k
0kto
_top10HCD
D1#8
_1#8713 RT: 555
5.56 AV: 1 NL: 1
116
.16E5
T: FTMS+pNSIdFull [email protected][100.00-1220.00]
408.2104
2
3
2
.13
9
8
100
900
1000
1100
1200
209.1027
1300
1400
200
300
400
20
200
815.4147
860.4276 912
122
.2214
745.4022
0
100
403.2353
300.1898
918.5521
790.3936
1438.2389 1593.8347 1679.2257 1853.9678
1024.0963 1162.3309
400
600
800
1000
m/z
1200
1400
1600
500
600
700
800
900
1046.2
62002
1000
40
20
1100
0
100
1200
200
300
400
1042.5245
817.4120
567.2697 680.3534
500
600
505.2741
CID
910.4715
175.1186
7344
4.4208
200
300
400
500
600
700
1009.5
95458 1124.5
45596
800
900
1000
1100
B
1256
569
.9731
1200
1300
1400
700
800
m/z
1143.5685
945.4672
900
m/z
1000
1100
1256
566
.6429
1200
14457
5.7957
1300
1400
1500
80
40
175.1185
591.2900
290.1455
690.3584
389.2133 520.2537
4691
9.1678
20
0
100
1600
200
300
400
500
600
924.4564
837.4249
1110.5138
965
540
.4013
700
800
900
m/z
1000
1100
6000
1224
454
.5483
1200
1400
5858
1500
5000
3000
2000
100 ng
1 µg
100 ng
0
9
10
0
CID
A
B
Protein ID TOP20 CID, 60 min gradient
CID
proteins
peptides
proteins ≥ 2peptides
Unique peptides (1 µg)
C
Unique proteins (1 µg)
Peptide ID TOP20 CID, 60 min gradient
12000
1050
HCD
FIGURE 6
6. Venn diagram comparing single ddTOP20 CID and
ddTOP10 HCD runs acquired
i d ffrom 1 µg and
d 100 ng E.
E coli
li digest
di
t
with a 90 min gradient ((2%-30%
% % B)) based on proteins ((A)) and ((B))
and unique
q p
peptides
p
((C)) and ((D)) identified.
FIGURE 3. Unique proteins (A) and unique peptides (B) identified
at <1% FDR from different sample amounts: a proteolytic digest
of 1 µg, 100 ng,
g, and 20 ng
g E. coli whole cell lysate,
y
, analyzed
y
with a
ddTOP20
dd
O 0 CID
C method
et od (2%-30%
( % 30% ACN
C in 60 min).
)
A
1 µg
1000
10000
HCD
184
unique
q
p
peptides
p
984
1 µg
CID
HCD
1861
53
5775
868
9545
945
8000
844
799
686
600
6582
6000
6468
572
5339
400
4000
4224
0
100 ng
B
0
20 ng
11µg
100 ng
CID
FIGURE 4. NanoLC-MS/MS analysis
y
of 1 µg E. coli digest
g
using
g
TOP10 HCD method (1x
( OT full scan + 10x ddHCD scans).
)
A) Base peak chromatogram displaying the scan speed for a full
scan cycle
cycle. B) 10 consecutive HCD MS/MS spectra and their
assigned peptide sequences
sequences. C) Peptide ID from Protein Discover
based on the HCD spectra displayed in B)
B).
Re
elative Abund
dance
30.47
35.47
24.32
60
26.92
24.11
52.57
52.65
45.84
47.80
67.56
54.41
35.34
92.21
91.87
94.66
88.20
68.01
58.31
58.63 61.96
58.22
74.55
71 44
71.44
76.10
Base Peak
B
P k
MS
EColi_noIId
et_30k_top
10HCD 1
10HCD_1
92.11
74.17
67.90
74 32
74.32
74.41
74.07
52 73
52.73
41.88
30.19
40
45.74
41.98
30 89
30.89
30.31
67 85
67.85
67 66
67.66
50.23
45.45
94.81
79.08
79.31 83.08
17.13 17.84
97.77
106.05
106.63
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
RT: 53.376 - 53.497
1 OT Full MS scan + 10 x dd HCD scans
53.406
NL:
3.65E7
53.460
53.471
Base Peak
MS
EColi_noIId
et_30k_top
10HCD_1
3.24 sec
300
400
500
80
147.1123
60
40
600
700
501.2730
564.7686
53.473
53.426
53.419
53.416
53.393
53.398
53.389
53.430 53.435
53.444
53 38
53.38
53 39
53.39
53 40
53.40
53 41
53.41
53 42
53.42
53 43
53.43
53.453
53.484 53.488
53 44
53.44
53.496
53.480
53.448
53 45
53.45
53 46
53.46
53 47
53.47
53 48
53.48
53 49
53.49
Time (min)
EColi_noIIdet_30k_top10HCD_1 #8258 RT: 53.41 AV: 1 NL: 2.88E7
T: FTMS + p NSI F
T
Full
ll ms [380
[380.00-2000.00]
00 2000 00]
100
554.8397
z=2
551.3011
z=?
556.3442
z=2
Relative Ab
bundance
80
60
40
20
RT = 53.406 min; OT Full MS, 1e6, 30k
630.3369
z=2
200
300
400
500
600
700
800
m/z
448.7636
z=2
406.9616
406
9616 430.2322
z=?
z=4
495.6001
z=3
515.7930
z=2
479.7683
z=?
642.3696
z=2
806.3810
z=2
655.3430
z=2
580.2760
z=3
747.3893
z=3
788.8860
z=2
703.6799 742.8968
z=2
z=3
3
859.4209
z=2
2
932.5008
z=2
450
500
550
600
650
700
750
800
850
900
1400
1000
1100
1366.6
66221
1200
1300
1400
1500
744
48
.8801
207.1122
20
870.4299
618.6508
235.1070
332.1048
567.7501
400
926.9
69765
600
800
967.4793
967
4793
z=2
950
1000
m/z
1200
1400
1600
1800
2000
References
425.2243
60
265.9941
40
234.1442
279.0970
837.4692
623.3741
392.1806
213.1157
20
738.4023
460
003
.0355
701
013
.3510 756.3984
200
300
400
500
600
700
919
946
.4628
800
900
1347.3011
1000
1100
1200
1300
Rel ative Abun
ndance
80
60
40
221.0
091
9 6 336
361
.1184
200
300
400
500
600
792.3927 893.4396 1008.4644
700
900
1000
1100
1200
1300
1400
40
209.1027
251.1496
347.1664
20
200
300
414.2123
460.2502 546.2836
400
500
918.5521
790.3936
860.4276 912.2214
600
700
1
2
3
4
5
800
900
1046.2002
1000
80
400
600
800
1000
m/z
1200
1400
1600
40
1100
1200
200
300
400
1042.5245
800
03
.3905
500
600
700
910.4715
399.1949
175.1186
734.4208
200
300
400
500
600
700
1009.5458 1124.5596
800
900
1000
1100
1256.9731
1200
1300
1400
m/z
817.4120
800
7694
69.4191
100
40
0
100
505.2741
EColi_noIIdet_30k_top10HCD_1#8709 RT: 55.54 AV:1 NL:8.81E4
T: FTMS+pNSI dFull [email protected][100.00-1550.00]
60
567.2697 680.3534
171.1124
20
0
100
806
60
.0945
185
851
.1279
339
392
.2019
256.1649 357.2124
797.3891
286.1393
1800
80
20
561.2621
349.6637
60
1438.2389 1593.8347 1679.2257 1853.9678
1024.0963 1162.3309
100
81541
5.4147
m/z
C
200
678.3557
745.4022
0
100
403.2353
300.1898
20
1500
607.3185
249.1592
136.0753
201.1233
602.3527 685.3633
464.2565
945.4672
900
m/z
1000
1143.5685
1100
1256.6429
1200
1300
1445.7957
1400
1500
1600
1 W
1.
Wouters,
t
E
E.R.,
R S
Splendore,
l d
M
M., S
Senko,
k M
M.W.,
W S
Syka,
k JJ.E.P.,
EP D
Dunyach,
h
J J Design
J.-J.,
D i off a Progressively
P
i l Stacked
St k d Ring
Ri Ion
I Guide
G id for
f Improved
I
d
Ion Transmission at High Pressure, ASMS 2008.
647.8408
0
80
60
40
459.7770
173.1280
EColi_noIIdet_30k_top10HCD_1#8711 RT: 55.55 AV:1 NL:1.34E5
T: FTMS+pNSI dFull [email protected][100.00-1625.00]
EColi_noIIdet_30k_top10HCD_1#8713 RT: 55.56 AV: 1 NL: 1.16E5
T: FTMS+pNSI dFull [email protected][100.00-1220.00]
408
82
.2104
232.1398
100
Relative Abund
dance
800
m/z
1171.5236 1259.9012
80 120.0803
60
698.3206
100
Rela
ative Abund
dance
0
100
394.1754 528.2807 629.3283
Rela
ative Abund
dance
20
EColi_noIIdet_30k_top10HCD_1#8704 RT: 55.52 AV: 1 NL: 6.11E5
T: FTMS+pNSI dFull [email protected][100.00-1420.00]
Relative Abu
R
undance
Rela
ative Abundance
m/z
EColi_noIIdet_30k_top10HCD_1#8712 RT: 55.55 AV: 1 NL: 2.04E5
T: FTMS+pNSI dFull [email protected][100.00-1520.00]
193
309
.0967
100
p
provides
a higher
g
HCD scan speed
p
and much improved
p
spectral
p
quality,
q
y, opening
p
g up
p new perspectives
p p
for de novo sequencing,
q
g,
quantitation of chemically labeled samples, and analysis of
modifications which require high mass accuracy
accuracy.
80
0
100
EColi_noIIdet_30k_top10HCD_1#8710 RT: 55.54 AV:1 NL:2.12E4
T: FTMS+pNSI dFull [email protected][100.00-1870.00]
677.3086
100

1929.8
98708
1222
225
.5409
1000
m/z
EColi_noIIdet_30k_top10HCD_1#8707 RT: 55.53 AV: 1 NL: 9.72E4
T: FTMS+pNSI dFull [email protected][100.00-1395.00]
443.2350 542.3033
100
842.4172
z=2
812.4036
z=2
1300
729.0183
60
40
6542 peptides total
identifies more than 1,000
1 000 proteins per hour from the proteolytic
digest of an E.
E coli whole cell lysate
lysate.
1256.9731
1200
80
200
0
400
900
0
622.0023
z=3
610.6240
z=3
1100
EC
Cli
oli_noIIdet_30k_
k top1
10HCD
D1#
_1#87
8 06 RT: 55.53 AV: 1 NL: 2
23
.30E5
T: FTMS+pNSI dFull [email protected][100.00-2000.00]
120.0803
100
Relative A
Abundance
53.385
53.381
1000
724.4352 825.4767 876.4540
972.5574
759.3694
992.0
20388 1132
20
.0391
1.0
0.0
900
643.4118
416.1776
0
100
53.477
0.5
1009.5458 1124.5596
800
244.0922
329
91
.1811
20
1.5
Relative Ab
R
bundance
Relative Ab
bundance
2.5
2.0
734.4208
200
EColi_
li noIIdet_
t 30k_
k top10HCD
D1#
_1#8705 RT: 55
555
.53 AV: 1 NL: 1
12
.20E5
T: FTMS+pNSI dFull [email protected][100.00-1515.00]
129.1018
100
110
100 ng

910.4
04715
399.1949
m/z
Time (min)
3.0
505.2
52741
175.1186
0
100
0
15
797.3891
286
61
.1393
171.1124
20
773
provides a significantly increased scan speed in MS and MS/MS
mode
d due
d tto it
its hi
higher
h ttransmission
i i iion source, d
duall pressure ion
i
trap, and improved HCD cell.
561.2621
349.6637
60
111.83
95.03
88.05
80
40
4072

647.8408
111 91
111.91
94.92
698.3206
100
Relative Abundancce
45.60
30.57
80
NL:
2.24E8
Base Peak Chromatogram
g
1697
C
Conclusions
l i
ECo_
oli no
od
IIdet_
t 30_
0k top
p10HCD1
_ #8
8704 RT: 555
55.52 AV: 1 NL: 6.11E5
T: FTMS+pNSI dFull [email protected][100.00-1420.00]
Relative A
Abundance
e
67.71
41
HCD
The LTQ Orbitrap
p Velos hybrid
y
mass spectrometer
p
RT: 14.08 - 114.56
100
787
1059 proteins total
B
2%-30% ACN in 90 min
Unique peptides (100 ng)
CID
HCD
231
A
D
Unique proteins (100 ng)
20 ng
Raw data files were searched with the Mascot™ algorithm (Matrix
S i
Sciences)
) using
i Thermo
Th
Scientific
S i tifi Proteome
P t
Discoverer
Di
software
ft
v. 1.2.
12
20
8504 peptides total
1221 proteins total
3765
2000
11µg
While the first method uses the ion trap
p to detect the CID fragment
g
ions,,
the second method employs the Orbitrap to detect the HCD fragment
ions providing accurate masses for both MS and MS/MS scans
ions,
scans. In
addition to the different acquisition methods
methods, two different gradient
lengths were applied: 60 min and 90 min (2%
(2%-30%
30% B)
B). The methods and
the res
results
lts are ssummarized
mmari ed in Fig
Figures
res 2
2-6.
6 NanoLC
NanoLC-MS/MS
MS/MS analyses
anal ses of
1 µg E.
E colili digest
di
t with
ith a 60 min
i gradient
di t and
d a ddTOP20 CID method
th d
yields 1050 unique proteins based on 6582 unique peptides (results
obtained with an FDR < 1%).
)
4759
1
7
8
Data Processing
(1) TOP20
method (1 OT Full MS scan + 20 dd CID scans),
scans) and
(2) TOP10 HCD method (1 OT Full MS scan + 10 dd HCD scans).
scans)
6344
1376.9
69908
1300
2
3
4
5
200
ITMS2
7601
7000
274.1868
60
6
800
(2%
gradient
di
U i
Unique
P
Peptide
tid ID
IDs, 90 min
i gradeint
d i t (2%-30%
(2
(2-30
3030%
%B)B)
4000
C
1000
HCD
8000
769.4191
100
800.3905
100 ng
399.1949
EC
Cli
oli_noIIde
IId t_30k_top10HCD
D1#809
_1#8709 RT: 55.54 AV
V1
: 1 NL:8
L 881
.81E4
T: FTMS+pNSI dFull [email protected][100.00-1550.00]
40
185.1279 339.2019
256.1649 357.2124
286.1393
171.1124
m/z
60
1 µg
0
797.3891
60
1800
80
20
561.2621
349.6637
0
100
806.0945
100
414.2123
460.2502 546.2836
347.1664
201.1233
1500
607.3185
251.1496
40
53633
602
023
.3527 685.3
464.2565
100 ng
6478
7.8408
80
0
678.3557
249.1592
20
459.7770
1731
3.1280
EC
Cli
oli_noIIde
IId t_
t 30k
0kt
_top10HCD
D1#8
_1#8711 RT: 555
5.55 AV1
V: 1 NL:
L1
134
.34E5
T: FTMS+pNSIdFull [email protected][100.00-1625.00]
60
40
60
1171
15
.5236 1259
990
.9012
80
136.0753
80 120.0803
1 µg
200
698.3206
100
Relative Abundance
A
20
0
100
This complex peptide mixture was separated using a Thermo Scientific
S
f
Surveyor LC and MicroAS autosampler with a reversed-phase peptide
trap
p ((100 µ
µm inner diameter, 2 cm length)
g ) and a reversed-phase
p
analytical
y
column (75
( µm
µ inner diameter,, 10 cm length,
g , 3 µm
µ particle
p
size both NanoSeparations,
size,
NanoSeparations NL),
NL) at a flow rate of 300 nL/min.
nL/min
To evaluate the performance of the new linear ion trap
trap-Orbitrap
Orbitrap mass
spectrometer
t
t ffor characterization
h
t i ti off complex
l proteomes,
t
nanoLCLC
MS/MS analysis
l i off 1 µg, 100 ng, and
d 20 ng off a proteolytic
t l ti digest
di
t off
E. coli whole cell lysate was performed. Two different instrument
methods were employed for this analysis:
1017
995
800
425.2243
60
EColi_n
li noIIdet_
t 30k
0kto
_top10HCD
D1#8
_1#8712 RT: 55
55
.55 AV: 1 NL: 2
204
.04E5
T: FTMS+pNSIdFull [email protected][100.00-1520.00]
193.0967
100
1200
Results
1157
1000
200
80
0
100
Liquid Chromatography
FIGURE 1. Schematic of the LTQ Orbitrap
p Velos hybrid
y
mass
spectrometer equipped with an ETD source.
(2%-30%
(2%
30% B)
(2%-30%
Protein ID, 90 min gradient (2-30
%B)B)
80
200
0
400
500
EColi_noIIdet_30k_top10HCD_1#8707 RT: 55.53 AV: 1 NL: 9.72E4
T: FTMS+pNSIdFull [email protected][100.00-1395.00]
443.2350 542.3033
100
754.9254
z=2
683.3400
z=2
z
2
400
501.2730
147.1123
60
Relative Abundance
A
Relative Abundance
627.8640
z=2
40
300
0
724.3933
z=2
568.3077 617.8195
z=2
z=4
418.9117
z=3
200
80
RT = 43.856 min; OT Full MS, 1e6, 30k
80
60
910.4715
7344
4.4208
Relative Ab
bundance
43.826
505.2741
399.1949
175.1186
EColi_noIIdet_30k_top10HCD_1#8706 RT: 55.53 AV: 1 NL: 2.30E5
T: FTMS+pNSIdFull [email protected][100.00-2000.00]
120.0803
100
43.869
4
171.1124
20
0
100
6
2
Significant technology improvements have been implemented in the
LTQ Orbitrap
p Velos™ mass spectrometer,
p
including:
g (a)
( ) a progressive
p g
stacked-ring
g ion guide
g
(S-Lens)
(
) with 5-10x increased ion transmission;;
(b) a dual-pressure
dual pressure differentially pumped ion trap with a higher-pressure
higher pressure
cell for improved ion trapping,
trapping isolation,
isolation and CID efficiencies
efficiencies, and a
lower-pressure cell for improved resolution and/or scan speed; (c)
predictive AGC for increased scan speed; (d) an HCD-collision
HCD collision cell with
axial field for improved performance; (e) improved vacuum in the
O bit
Orbitrap
chamber
h b ffor b
better
tt iintact
t t protein
t i analysis.
l i
Methods
60
797.3891
286.1393
EColi_noIIdet_30k_top10HCD_1#8705 RT: 55.53 AV: 1 NL: 1.20E5
T: FTMS+pNSIdFull [email protected][100.00-1515.00]
129.1018
100
84.22
77.49
Time (min)
10
Enormous improvements in mass spectrometry technology over the last
few years have allowed for ever-deepening analysis of complex
proteomes However,
proteomes.
However many low-abundance
low abundance proteins remain undetected
in current large-scale
large scale proteomic analyses due to the limits on the rate at
which
hi h MS/MS spectra
t can be
b acquired.
i d To
T solve
l this
thi undersampling
d
li
problem, we developed a new hybrid linear ion trap-Orbitrap mass
spectrometer (Thermo Scientific LTQ Orbitrap Velos hybrid mass
spectrometer,
p
, Figure
g
1)) that p
provides faster MS/MS scanning
g in the ion
trap
p ((up
p to 10 p
peptide
p
sequences
q
p
per second),
), thereby
y increasing
g the
depth of analysis of complex protein mixtures. In the present work, we
evaluated the ability of the newly developed hybrid instrument to identify
more proteins,
proteins with increased sequence coverage and confidence
confidence.
55
40
Relative Ab
bundance
30
Relative A
Abundance
e
Introduction
25
561.2621
349.6637
60
m/z
0
20
80
0
100
69 66
69.66
64.84
20.94
19.01
647.8408
69.54
61.92
698.3206
100
Relative Ab
R
bundance
Purpose: Evaluate the performance of the newly developed hybrid
linear ion trap-Orbitrap
trap Orbitrap mass spectrometer for characterization of
complex proteomes
Methods: NanoLC-MS/MS analyses of a complex protein proteolytic
digest from E. coli whole cell lysate
FIGURE 2. NanoLC-MS/MS analysis
y
of 1 µg
g E. coli digest
g
using
g
TOP20 CID method ((1x OT full scan + 20x ddCID scans).
)
A) Base peak chromatogram displaying the scan speed for a full
scan cycle. B) 10 consecutive CID MS/MS spectra and their
assigned peptide sequences
sequences. C) Peptide ID from Protein Discover
based on the CID spectra displayed in B.
B
Relattive Abundan
nce
Overview
1 µg of E.
E coli digest was separated over 90 min and analyzed with CID
(ddTOP20 CID method) and HCD (ddTOP10 HCD method) in separate
runs The number of identified proteins was well above 1000 for each run,
runs.
run
based on 7601 and 6344 peptides
peptides, respectively
respectively, (Figure 5) as an average
f
from
d
duplicate
li t runs. Th
The overlap
l off id
identifications
tifi ti
ffrom single
i l runs using
i
C versus HCD
CID
C was 81% on the protein level and 68% on the peptide
level. The speed of CID and HCD MS/MS spectra acquisition is
significantly
g
y increased on the LTQ Orbitrap
p Velos hybrid
y
mass
spectrometer,
p
, which is mainlyy due to the much reduced fill times.
80
274
741
.1868
60
40
175.1185
20
0
100
200
591.2900
290.1455
690.3584
389.2133 520.2537
469.1678
300
400
500
600
700
2 Pekar Second
2.
Second, T
T., Blethrow
Blethrow, JJ.D.,
D Schwartz
Schwartz, JJ.C.,
C Merrihew
Merrihew, JJ.E.,
E
MacCoss M.J.,
MacCoss,
M J Swaney,
Swaney D.L.,
D L Russell,
Russell JJ.D.,
D Coon
Coon, JJ.J.,
J and
Z b
Zabrouskov,
k
V.
V Dual-Pressure
D lP
Li
Linear IIon T
Trap M
Mass Spectrometer
S
t
t
I
Improving
i the
h Analysis
A l i off Complex
C
l Protein
P
i Mixtures,
Mi
A l Chem.
Anal.
Ch
2009, 81, 7757–7765.
924
445
.4564
837.4249
965.4013
800
900
m/z
1000
1110.5138
1100
1224.5483
1200
1376.9908
1300
1400
1500
3 Olsen JJ.V.,
3.
V Schwartz JJ.C.,
C Griep-Raming
Griep Raming J.,
J Nielsen M
M.L.,
L Damoc
Damoc,
E D
E.,
Denisov,
i
E
E., L
Lange, O
O., R
Remes, P
P., T
Taylor,
l D
D., S
Splendore,
l d
M
M.,
W t
Wouters,
E.R.,
ER S
Senko,
k M.,
M Makarov,
M k
A.,
A Mann,
M
M.,
M Horning,
H i
S.
S AD
Duall
Pressure Linear Ion Trap - Orbitrap Instrument with Very High
Sequencing Speed. Mol Cell Proteomics. 2009 Dec; 8(12):2759-69.
6
7
8
9
10
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