Mass Spectrometry Facility Advanced Analysis Centre University of Guelph Presented by: Dr. Dyanne Brewer Mass Spectrometry Facility Manager May 2014.

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: [email protected]: x58649 lab x53793 DB office