High throughput accurate mass measurement using the LDTD ion source on the LTQ Orbitrap Faubert D.1, Venne K.2, Champagne C.1, Boulos M.1, Furtos A.2, Letarte S.3, Picard P.3 and Coulombe B.1 1. Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Canada 2. Centre Régional de Spectrométrie de Masse, Université de Montréal, Canada 3. Phytronix Technologies, Québec, Canada Table 1 – Relative intensity (RI) of various molecules analysed with ESI, APCI and LDTD ion sources Sample
ESI
RI (%)
APCI
RI (%)
LDTD
[M+H]+ [M+H]+
100 100
100 100
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
Accurate mass measurements (AMMs) of small molecules by mass spectrometry were mostly performed on magnetic sector instruments until the end of the last century. Although these instruments provide sufficient accuracy/resolution for AMMs, they suffer from important limitations compared to new generations of high resolution instruments. Sector instruments are difficult to tune and to operate, their throughput is low and reference compounds increase the complexity of spectra. ESI/APCI sources have revolutionized the analysis of small molecules by LC/MS and for AMMs. Nevertheless, these sources present their own drawbacks like throughput limited to less than 30 samples/hour, high solvent consumption, requirement of an autosampler and LC pump.
[M+H]+ [M+H]+
150 148
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 100 100 100
100 100 100 11 100
[M+H]+ [M+H]+
23 36
10 30
042 043 [M+H]+ [M+H]+
[M+H] [M+H]+
40 34
[M+H]+ [M+H]+
159 160
[M+H]+ [M+H]+
19 80
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 62 100 100 100
[M+H]+ [M+H]+
2 100
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 100 100 43
+
102 103 104
[M+H] [M+H]+ [M+H]+
+
100 100 46
[M+H] [M+H]+ [M+H]+
100 100 24
17/05/2007 14:53:49
[M+H] [M+H]+ [M+H]+
1
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
015 049 054 106 107 108
RI
LDTD
RI
[M+H] [M+H]+
+
96 72
[M+H] [M+H]+
100 100
037 038 039
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 100 100 5
+
[M+H] [M+H]+ [M+H]+
+
[M+H] [M+H]+ [M+H]+
100 7 100
+
14 8 100
[M+H] [M+H]+ [M+H]+
100 8 78
Sample
ESI
RI
APCI
RI
LDTD
Table 2 – Efficiency of ESI, APCI and LDTD for accurate mass measurements (AMMs) of small molecules.
RI
+
085 092 099 181
[M+H] [M+H]+ [M+H]+ [M+H]+
+
58 100 97 100
[M+H] [M+H]+ [M+H]+ [M+H]+
+
100 100 100 28
[M+H] [M+H]+ [M+H]+ [M+H]+
Number of samples Number of identification Specificity of each source Success rate for AMM
100 100 100 26
48 100 36 38 58 100
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 100 100 100
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 38 37 100 100
004 007
[M+H]+ [M+H]+
[M+H]+ [M+H]+
71 88
[M+H]+ [M+H]+
35 40
100 100
86 100 47 100 16 100
[M+H] [M+H]+ [M+H]+
74 100 100
+
+
063 064 065 067
[M+NH4] [M+NH4]+ [M+NH4]+ [M+NH4]+
024 026 136
[M+H] [M+H]+ [M+H]+
+
[M+H] [M+H]+ [M+H]+
31 100 100
+
+
100 100 100 100
1 100 100
[M+H] [M+H]+ [M+H]+
2 100 100
+
028 144
[M+H] [M+Na]+
[M+NH4] [M+NH4]+ [M+NH4]+ [M+NH4]+
100 18 35 96
+
[M+NH4] [M+NH4]+ [M+NH4]+ [M+NH4]+
Sample
ESI
RI
[M+H] [M+H]+ [M+H]+
100 8 11
[M+H] [M+H]+ [M+H]+
84-215 87 95-98 96-188 180 185-187 187
[M+H]+ [M+3H]3+ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+H]+ [M+H]+
37-62 100 5-74 100 100 24-16 16
+
15 12
[M+H] [M+H]+
ND 9
+
[M+H] [M+H]+
[M+H]+ [M+H]+ [M+H]+ [M+H]+
100 100 22
100 100 57 80
100 100 100
[M+H]+ [M+H]+
[M+H]+ [M+H]+
6 35
ND ND
[M+H]+ [M+H]+
[M+H]+
218 090 091
+
[M+H] [M+H]+
+
[M+H] [M+H]+
100 100
+
100 100
[M+H] [M+H]+
[M+H]+
ND
ND
[M+H]+
100
201-204
100 100
0.12
70 60 50 40
80
60
10 0 0.10
0.15
0.19 0.02 0.10
Relative Abundance
Relative Abundance
[2M+H]+
30
0 100
200
564.97180 C30 H17 N2 79Br 1 81Br 1 -2.56577 ppm
373.04559 C22 H16 N1 79Br 1 -1.25732 ppm 300
0.25
887.01495 C46 H25 N4 127I2 -1.54895 ppm
16/05/2007 14:00:46
400
500 m/z
600
100 100 62 9 100 100
1
[M+H]+ [M+H]+ [M+H]+
221 222 223
150 141 8 94 %
APCI [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
RI
LDTD
RI
(not included in Table 1 and 2) ND [M+H]+ ND ND [M+H]+ ND ND [M+H]+ ND ND [M+H]+ ND 5 [M+H]+ ND + ND [M+H] ND 48 [M+H]+ ND
[M+H]+ [M-HCl]+
16 100
[M+H]+ [M-HCl]+
0.06 100
[M+H]+ [M-HCl]+
0.12 100
[M+H]+ [M-Boc]+ [M+H]+ [M-Boc]+
58 100 5 100
[M+H]+ [M-Boc]+ [M+H]+ [M-Boc]+
0.1 100 0.1 100
[M+H]+ [M-Boc]+ [M+H]+ [M-Boc]+
0.4 100 0.3 100
[M+H] [M+Na]+
[M+H]+ [M+H]+ [M+H]+
ND ND ND
ND ND ND
[M+H]+ [M+H]+ [M+H]+
66 20 100
CONCLUSIONS
[M+H]+
192
[M+H]+
ND
ND
• Highest throughput for accurate mass measurements (300 samples/hour) • Success rate for AMMs comparable to ESI and better than APCI • LDTD provides better spectral quality than ESI and APCI for most compounds • Specificity for the analysis of polyaromatics and other classes of molecules containing few functional groups
[M+H]+
3 100
+
[M+H] [M+Na]+
+
ND 1
[M+H]
Limitation • Poor efficiency for the analysis of thermolabile biomolecules such as peptides The coupling of the LDTD ion source with LTQ Orbitrap hybrid mass spectrometer is the ideal solution for high throughput AMMs of small molecules.
ND
ACKNOWLEGMENTS [M+Na]+ [M-H2O+H] + [M+H]+
125 95
126
76 66 100
[M+NH4]+ [M-H2O+H] + [M+Na]+
[M+H]+ [M-H2O+H] + [M+NH4]+
7 17 8
The authors thank Dr Yvan Guindon, Dr Peter W. Schiller, Dr Daniel Chapdelaine, Dr James D. Wuest and their teams for providing the samples and allowing us to present the molecular structure of some samples.
0.25 100 0.7
Throughput achievable with the LDTD source
Figure 3 – Cont’d LDTD20070516_HZ-01-160_500nL_18%
217.11
16/05/2007 14:06:38
1
D:\ASMS_2007\ESI\LO_20070504_MB-198-p078
40
439.19 529.23
80 60 40
[M+H]+
217.11
619.28
394.00
20 453.17
1180.85 1285.77
LDTD
[M+H]+
04/05/2007 14:21:52
Figure 4 – Multiple samples chromatogram obtained with the LDTD source
393.99
40 568.85
191.86 0 100
0 100
273.94
APCI
[M+H]+ 80 60 217.11 148.04 290.96
20
302.96 0 100
273.94
LDTD
[M+H]+ 80
80
764.99
60
192.30
80 60 40
0 100
0.30
529.23
40
761.11743 C46 H26 N4 127I1 -2.94824 ppm
40 201.05716
373.04544 304.98123
489.13266
60
644.97 743.01
372.01
LDTD
[M+H]+ 80
331.10
0 100
417.21
[M+H]+ 599.20
925.35
331.10
0.50
#194
APCI
80
60
#26
50
0.70
1.32
40
40
1.73
10
[M+NH4]+
40
#41 #100
20
211.06
0 100
417.21 431.18
20
20
338.34 718.39
0 200
400
600
800 m/z
200
300
400
500
600 m/z
700
800
900
1000
1100
919.39 1049.94 1237.56 1000
1200
20
219.96
391.28 0 100
743.01
0 200
300
400
500 m/z
600
700
800
200
400
600 m/z
800
2.33
1.14
BLK
0 0.0
331.10
0.2
1.55
0.75
0.27 0.4
0.6
0.8
1.0
1.2
[M-H]+ 923.33 1024.45
0
417.21 507.25 400
1.8
2.0
2.2
2.4
#174 2.53 2.55 2.6
2.81
2.8
3.0
655.23 600
849.40 800
1125.43
m/z
1000
1200
Throughput obtained with one sample per raw file
Sample #113
[M+H]+
≤ 26 seconds (sample-tosample)
70 60 50
Functional groups : Br, CO2Me, OBn, TBDPSO, OSi(R)2CHCH2
40 293.02078 C 11 H 16 O 4 81 Br 1 0.62016 ppm
30 20
200
1.6 Time (min)
551.14734 C 26 H 38 O 4 81 Br 1 28Si 2 1.32542 ppm
100
80
211.06
1.4
2.36
1.95
T: FTMS + p ESI Full ms [ 80.00-1600.00]
LDTD
80
20
226.96
#94
2.13
954.37 1024.45
40 287.95
1.93
#113 0.07
942.38
169.05
40
#42
1.12
20
169.05
≤ 12 seconds (sample-to-sample)
2.78
#31 #43 0.91
1.54
60
Throughput obtained with multiple samples per raw file
#153
30
60
354.00
493.24
184.99
635.22076 C46 H27 N4 -3.55699 ppm
60
2.77
Sample # on peak top
70
169.05
796.98 895.07
APCI
354.00 389.04
#27 #22
2.97
[M+H]+
199.10
[M+100]+
40
372.01
20
100
80
743.01 653.28
#21
RT: 0.00 - 3.10
90
20
354.00
942.38
[M+NH4]+
1024.45
471.11
20
765.00
666.93
[2M+Na]+ [2M+H]+
[M+100]+
ESI
100
90
60
50
0 100
918.59
641.26 619.28
[M+Na]+
60
20
40
[M+H]+
80
40
1259.54 1337.67 947.90 1051.01
APCI
60
30
736.40
ESI
372.01
100
295.92
60
[2M+H]+ [2M+Na]+
ESI
372.01
80
80
70
10 800
[M+100]+
529.23
0 100
[M+H]+
20
700
273.94
100
60
249.13
80
40
201.05722 C15 H7 N1 -0.39993 ppm
0.20
90
50
10
0.15 Time (min)
100
60
80
0 100
LDTD20070517_se-e73-tetracyanodiiodo_2uL_18% #18 RT: 0.16 AV: 1 NL: 1.18E7 T: FTMS + p ESI Full ms [ 100.00-1700.00]
[M+H]+
[M+100]+
619.28
282.28
0.05
70
20
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
64 100 4 2 100 100
471.12
0.20
LDTD20070517_se-e84-cyanobromidetolan_500nl_18% #13 RT: 0.12 AV: 1 NL: 3.72E8 T: FTMS + p ESI Full ms [ 100.00-1700.00] 281.99081 C15 H9 N1 79Br 1 -1.67676 ppm 100
ESI
718.39
[M+H]+
20
Time (min)
80
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
40 100 30 10 100 100
LDTD20070516_HZ-01-103A_500nL_25%
31/01/2007 15:05:58
100
40
10
All ion sources were operated in positive ion mode without any method development for each sample. Standard parameters were used for all three sources. The laser power energy of the LDTD source was set to 18 or 25%.
[M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+
50
20
0
0.13
70
30
0.05
NL: 2.08E7 m/z= 886.16-889.82 MS LDTD20070517_ se-e73tetracyanodiiodo _2uL_18%
90
20
90
100 100 100 84
LO_20070131_bcd_202_p101_f1
1
0.15
100
30
Instrumental parameters:
034 130 135 137 027 134
Figure 3 – Examples of spectra obtained with LDTD in comparison to ESI and APCI 17/05/2007 15:20:49
150 134 0 89 %
100 100
Relative Abundance
Relative Abundance
Nozzle Piston
[M+H]+ [M+H]+ [M+H]+ [M+H]+
NA 100 26 66
Relative Abundance
NL: 9.15E8 m/z= 281.60-285.46 MS LDTD20070517_s e-e84cyanobromidetola n_500nl_18%
80
Transfer Tube
NA [M+H]+ [M+H]+ [M+H]+
150 142 5+4 95 %
The LTQ Orbitrap is ideally suited for accurate mass measurements using the LDTD ion source because mass accuracy lower than 3 ppm is obtained with external calibration. This avoids adding a reference compound to the sample for AMMs. The mass error range varied from 0.1 to 3 ppm for all analysis and was not affected by the ion source mounted on the LTQ Orbitrap. In terms of spectral quality, the LDTD spectra are cleaner than ESI and APCI spectra for most compounds (see Figure 3). Overall, the success rate of the LDTD source for detecting small molecules is comparable to ESI and slightly better than APCI (see Tables 1 and 2). Moreover, the LDTD source shows a unique specificity for the analysis of polyaromatic compounds over ESI and APCI (see Figure 2) and other classes of molecules (see Table 2). The only limitation of the LDTD source, observed so far, is its poor efficiency for the analysis of thermolabile biomolecules such as peptides and few cases of small molecules (see Table 3). Thermal fragmentation must occur before vaporization of the labile biomolecules. The sampling rate obtained with the LDTD source on the LTQ Orbitrap is approximately 300 samples per hour (Figure 4) while the best reachable rate with ESI and APCI is less than 30 samples per hour.
4 5
+
119 122 123 124
LDTD
Peptides
16 2 4 15
025 072 076
+
APCI
Strenghts of the LDTD source +
Functional groups : OBn, OH
+
ESI
DISCUSSION
Table 3 – Molecules for which no molecular ion of significant relative intensity were detected with LDTD
R
LDTD20070517_se-e73-tetracyanodiiodo_...
90
Carrier Gas Line
(ThermoFisher) (ThermoFisher) (Phytronix) (ThermoFisher)
APCI
RT: 0.01 - 0.26
100
Mobile phase and flowrate: ESI 60%MeOH – 0.1%FA, flowrate: 20 µL/min APCI 60%MeOH – 0.1%FA, flowrate: 300 µL/min LDTD no mobile phase, carrier gaz: N2 (99.998%) Mass spectrometer: LTQ Orbitrap (operated at RP 30,000)
RI
8 38
+
Relative Abundance
LazWell Sample Plate
Sample state: SOLID Thermal desorption is induced indirectly by laser at 980 nm (infrared). Sample is transferred by the carrier gas. The ionization is carried out by a corona discharge (constant voltage).
ESI
Functional groups : Br, I, OBn, OH, aldehyde
54 20
[M+H]+ [M+H]+
RT: 0.02 - 0.24
Corona Discharge
Sample
045
LDTD20070517_se-e84-cyanobromidetolan...
(Mass Spectrometer)
RI
Functional groups : Br, CO2Me, OH, OBn, TES, TBDPSO, OTBS, OSi(R)2CHCH2
Figure 2 – Molecules solely detected with LDTD
LTQ Orbitrap
+
100 100
20 21 22
The potential of the LDTD ion source for AMMs of organic molecules is demonstrated using hundreds of natural and synthetic compounds.
IR Laser Beam
LDTD
200
The present work involves the coupling of two new technologies for performing very high throughput AMMs. The LDTD (Laser Diode Thermal Desorption) source (Phytronix Technologies) has been mounted on the LTQ Orbitrap mass spectrometer (ThermoFisher Scientific). The LDTD technology consists of a rapid desorption of a dried sample followed by atmospheric pressure chemical ionization without solvent. The thermal desorption is produced by an infrared laser diode beam focused at the back of each well of a specially designed 96-well plate. The LTQ Orbitrap is a novel instrument combining all the features of the LTQ linear ion trap with those of the Orbitrap: a high scan rate mass analyzer, high mass accuracy (< 5 ppm, external calibration), and high resolving power (up to 100,000). All analyses were performed on the LTQ Orbitrap.
Figure 1 – The LDTD ion source
RI
Relative Abundance
INTRODUCTION
• • • •
+
051 052 053 055 113 154 155 156 013 158
APCI
Relative Abundance
Results – LDTD provides better performances than ESI and APCI in terms of throughput, ease of use, spectral quality and specificity for polyaromatic molecule detection. In terms of sensitivity and success rate for AMMs, LDTD is comparable to ESI and APCI. Overall results show that the LDTD ion source mounted on the LTQ Orbitrap is ideally suited for AMMs of small molecules within a MS core facility.
METHODS
[M+H]+ [M+H]+
093 097
RI
RI (%)
OC(CH3)3
Methods – Analysis of 150 natural and synthetic compounds using ESI, APCI and LDTD in positive ion mode.
ESI
Relative Abundance
Purpose - Evaluate the performance of the LDTD ion source in positive ion mode on the LTQ Orbitrap mass spectrometer for accurate mass measurements (AMMs) of small molecules.
Sample
Relative Abundance
OVERVIEW
10 0 100
200
300
400
500
600
700 m/z
800
900
1000
1100
1200
