mass spectrometry facility advanced analysis centre university of guelph presented by: dr. dyanne...
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Mass Spectrometry Facility
Advanced Analysis Centre University of Guelph
Presented by: Dr. Dyanne Brewer
Mass Spectrometry Facility ManagerMay 2014
MS Sample Submission Workflow
Data Interpretation
Sample Submission
Sample Preparation
Data Acquisition
Training Available:GC-MS derivitizationProtein digestion
Training Available:GC-MS LC-MSn
Training Available:Metabolomics softwareProteomics software
Submission Forms:http://www.uoguelph.ca/~bmsf/
Instrumentation
Agilent GC-MS Bruker GC-MS-MSSolid, liquid, gas, SPME
Bruker LC-MSn Agilent LC-HRMS-MS
GC- gas chromatographyLC- liquid chromatographyMS- mass spectrometry
Sample Type: Small Molecule
Small Molecule
Volatile/semi-volatile
GC separation
MS confirmation
MS-MS quantitation
Non-volatile LC Separation
MS confirmation/formula
MS-MS characterize/quantitate
Small Molecule GC-MS
Sample Collection Metabolite Extraction Compound Derivitization
7.5 10.0 12.5 15.0 17.5 20.0minutes
0.0
2.5
5.0
7.5
10.0
GC
ps
TIC; KIT_051964_T120_4-4-2013_Metabol2.xms; Filtered
100 200 300 400 500 600m/zR.Match: 845, F.Match: 819
0%
25%
50%
75%
100%
Search
59.6 7.291e+7
73.1 4.742e+8
147.1 1.494e+8
174.2 5.251e+8
248.2 1.042e+8
8.744 min, Scan: 1089
0%
25%
50%
75%
100%
Match
73.0 510
86.0 132
147.0 208
174.0 999
248.0 163
NSiSi
O
O
Si
Match 1 of 12
NIST match to Glycine N,N-bis(trimethylsilyl) trimethyl ester
Gas Chromatogram
Dr. Mutch UoG HHNS
Small Molecule Mass Accuracy1x10
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
C49 H54 Cl5 N8 O9 P: +ESI Scan (1.29-1.55 min, 33 Scans) Frag=170.0V Nov_13_AW_2.d
1107.2252([C49 H54 Cl5 N8 O9 P]+H)+
Counts (%) vs. Mass-to-Charge (m/z)1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127
Best ID Source Formula Species m/z Score Diff (ppm) Score (MFG)
TRUE MFG C49 H54 Cl5 N8 O9 P (M+H)+ 1105.2269 94.53 -0.33 94.53
Dr. Manderville UoG Chem
Small Molecule LC-MS
0 2 4 6 8 10 12 14 16 Time [min]0.0
0.5
1.0
1.5
2.0
2.5
8x10Intens.
Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 271.0 +All MS2 Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 449.0 +All MS2Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 727.0 +All MS Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 301.0 +All MS2Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 303.0 +All MS2 Cherry-profile-Jan-21-2014_BD2_01_1145.d: EIC 317.0 +All MS2Cherry-profile-Jan-21-2014_BD2_01_1145.d: Base Peak UV Chromatogram, 520 nm
UV 520 nmPelargonidin 271Cyanidin 287Peonidin 301Delphinidn 303Malvidin 331Petunidin 317
301.0
463.0
+MS2(609.4), 10.2min #571
0
20
40
60
80
100
Intens.[%]
200 400 600 800 1000 1200 1400 m/z
-peonidin
-hexose
-deoxihexose
Dr. Kirakosyan Un. Michigan
Small Molecule LC-MS
HeartBladderkidneyBrain
0 1000 2000 30000
50000010000001500000200000025000003000000
f(x) = 1017.3714004 x + 20978.236278R² = 0.99002029790023
Calibration Curve of Peonidin 3-O-glucoside
Tissue Extract
sample name RT [min] Area concentration_peonidin dihexoside_fg/ul
1%heart_1.54g_(5) 10.2 42548 32.6
1%Bladder_0.75g_(1) 10.2 12946 3.2
1%kidney_1.4g_(14) 10.2 91933 81.7
1%brain_0.94g-(9) 10.2 14337 4.6
Dr. Kirakosyan Un. Michigan
Compareand Stats
MS database search targets
Pathway?
Target MS/MS
Separate and Detect MS
Compare to database/standard
ID
MassHunter/MPP software
GC-MS/LC-MS
In-house Metlin/NIST
library or online
GC-MS data
Metabolomics Workflow
Protein
Intact characterization Clean-up LC MS
confirmation
Identification
Solution digest
In-gel digest
LC Auto MS-MS
Database search/de
novo sequencing
PTM id
Sample Type: Large Biomolecule
Protein MW
2x10
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
1.15+ESI Scan (10.69 min) Frag=300.0V ASR1-Rachel-Dec01.d
1607.4963
2276.8410
2101.7818
905.4612
724.57132483.7757
603.8107
364.1687 2732.16042921.9034
Counts (%) vs. Mass-to-Charge (m/z)400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200
6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2+ESI Scan (10.69 min) Frag=300.0V ASR1-Rachel-Dec01.d Deconvoluted (Isotope Width=11.0)
27
31
0.4
65
6
13
65
6.0
09
5
20
53
7.8
78
2
21
53
8.1
52
3
43
07
6.9
74
5
41
07
4.6
62
3
44
42
4.0
65
2
42
13
6.6
35
4
48
25
0.1
98
6
32
31
5.6
10
3
29
14
8.7
63
2
45
81
4.6
57
8
49
81
5.7
42
1
34
97
3.5
00
5
46
62
7.6
57
7
39
88
5.5
71
8
38
57
9.8
79
0
37
39
6.1
32
9
34
11
6.4
80
3
30
15
0.6
71
3
Counts vs. Deconvoluted Mass (amu)10000 12500 15000 17500 20000 22500 25000 27500 30000 32500 35000 37500 40000 42500 45000 47500 50000
Raw data
27,310.4656 amu
Protein solution Clean-up LC
Deconvoluted data
Dr. Brown UoG Physics
Protein Identification
Protein Group Protein ID Accession -10lgP
Coverage (%) #Peptides #Unique PTM Avg. Mass
Description
1 6181Colicin_E1
_Q372C 283.51 69 60 60 N 21734Colicin_E1
_Q372C
Protein digest
LC-Chromatogram
Peptide Fragmentation
Protein IdentificationDr. Merrill UoG MCB
Mass Spectrometry Facility
Advanced Analysis Centre University of Guelph
Manager: Dr. Dyanne Brewer Research Associate: Dr. Armen CharchoglyanLocation: Science Complex Rm 1205Email: dbrewer@uoguelph.caPhone: x58649 lab x53793 DB office
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