Relative and Absolute Quantitation of Metabolites and Lipids using LC/MS/MS on the TSQ ...

182 Pages·2016·2.24 MB·English

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Masters Theses Graduate School
5-2013
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Jesse Lee Middleton
University of Tennessee - Knoxville, [email protected]
Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes
Part of the Analytical Chemistry Commons
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Middleton, Jesse Lee, "Relative and Absolute Quantitation of Metabolites and Lipids using LC/MS/MS on
the TSQ Quantum Discovery MAX. " Master's Thesis, University of Tennessee, 2013.
https://trace.tennessee.edu/utk_gradthes/1649
This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and
Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE:
Tennessee Research and Creative Exchange. For more information, please contact [email protected].
To the Graduate Council:
I am submitting herewith a thesis written by Jesse Lee Middleton entitled "Relative and Absolute
Quantitation of Metabolites and Lipids using LC/MS/MS on the TSQ Quantum Discovery MAX." I
have examined the final electronic copy of this thesis for form and content and recommend that
it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with
a major in Chemistry.
Shawn R. Campagna, Major Professor
We have read this thesis and recommend its acceptance:
Michael J. Sepaniak, Robert N. Compton
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
Relative and Absolute Quantitation of Metabolites
and Lipids using LC/MS/MS on the
TSQ Quantum Discovery MAX

A Thesis Presented for the
Master of Science
Degree
The University of Tennessee, Knoxville

Jesse Lee Middleton
May 2013
Dedication
For current and future UT graduate students who want to learn LC/MS/MS

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Abstract
Two biological systems were studied using LC/ESI/MS/MS on a triple quadrupole operated in
SRM (selected reaction monitoring) scan mode. The first bacterium system is aquatic and
microscopic in size known as Roseobacter. The second mammalian system is terrestrial and
large in size relative to humans known as Holstein cows.
Roseobacter is a clade of marine bacteria abundant in the ocean. Roseophages are viruses that
infect Roseobacter and cause viral lysis. Sulfitobacter sp. 2047 was isolated and infected with
Roseophages, and the fold change in the metabolic pool relative to a control was studied at
discrete time points. The absolute concentration of glutamate and glutamine in the infected and
control was determined at each time point using an external calibration curve. Flux analysis
through the addition of 13C-acetate at early and late post infection was compared to the control.
Holstein cows are a breed of cattle known to be the world’s highest producers of milk. Twelve
Holstein dairy cows were selected, and samples of blood and milk were taken at different weeks
of lactation. The fold change in the phospholipid pool relative to the first week of lactation was
studied from early, mid, and late lactation. The absolute concentration of lipids at each week of
lactation was determined using isotope dilution mass spectrometry with the exception of GPC
(glycerophosphocholine) where an external calibration curve was used due commercial
unavailability of an isotope-labeled standard.

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Table of Contents
Introduction …...…………………………………………………………………………………. 1
Chapter 1
Metabolic Response of a Roseobacter to Phage Infection: Insights into the Influence of Viral
Lysis on Ocean Biogeochemistry ...………………………………………………………...…… 2
Abstract ...……………………………………………………………………………………...… 3
Background and Significance ...…………………………………………………………………. 4
Introduction ………………………....………………………………………………...……… 4
Roseobacter Biology …………………………...…………………………………………….. 5
Roseophage …………………………………………….…………………………………….. 6
Molecular Tools and Marine Virus Communities ………………………………...…………. 7
Merits of Liquid Chromatography-Mass Spectrometry ……………………………...………. 8
Triple Stage Quadrupole (TSQ) Quantum Discovery MAX ……………………………….. 10
High Performance Liquid Chromatography and Tandem Mass Spectrometry Method Transfer
Routine ……………………………………………………………...………………………. 14
Optimization of Chromatography ………………………………………………………..…. 14
Tuning and Calibration ………………………………………………………………...…… 21
Optimization of Ion Source Parameters at LC Flow ………………………………...……… 22
SRM Optimization ………………………………………………………………………….. 23
Alternative Methods to Remove SRM Interference without Mass Filtering …………..…… 33
Absolute Quantitation using External Calibration Curves ………………………………….. 34
Data Processing …………………………………………………………………...………… 35
Isotope Labeled SRM Transitions of 13C Flux Experiment ………………………………... 36
Results and Discussion ………………………………………………………………………… 37
Composition of Extracellular Small Molecule Components following Phage-Induced Cell
Lysis ………………………………………………………………………………………… 37
Alterations in Intracellular Metabolite Pools and Flux during Phage Infection ………….… 40
External Calibration Curves of Glutamine and Glutamate ……………………………….… 47
Calculations ………………………………………………………………….……………… 50
Methods and Materials ………………………………………………………………………..... 51
General Methods …………………………………………….……………………………… 51
Isolation of Bacterial and Phage Strains …………………………………………………..... 51
Culture Conditions and Sample Collection ……………………………………………...….. 52
Flux Analysis …………………………………………………………………….…………. 53
Extraction Procedure ……………………………………………………………...………… 53
Chromatographic Details …………………………………………………………………… 54
Mass Spectrometric Detection Parameters ………………………………………………….. 55
Data Processing …………………………………………………………..…………………. 55
Sample Matrix for External Standards ………………………………………...……………. 56
Data Processing for the Calculation of Absolute Quantitation using External Standard ….... 57
Chapter 2
Changes in Choline Esters in Blood and Milk during Early, Mid, and Late Lactation in Dairy
Cows …………………………………………………………………………………………… 59
Abstract ………………………………………………………………………………………… 60
Background and Significance ………………………………………………………………….. 61
Introduction ……………………………………………………………..……………………61
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Internal Standard ………………………………………………………………….………… 64
Results and Discussion …………………………………………………………..…………….. 68
Determination of Heatmap Log Scale ………………………………………...…………….. 68
Changes in Milk and Plasma at Different Weeks of Lactation Relative to Week 1 ……...… 69
Extraction Reproducibility of Milk and Plasma Samples ……………………….………….. 72
External Calibration Curve of GPC …………………………………...……………………. 75
Internal Standard SRM Determination …………………………...………………………… 75
Calculations ……………………………………………………………….………………… 78
Methods and Materials ………………………………………………………...……………….. 79
General Methods ……………………………………………………………………………. 79
Extraction Procedure ……………………………………………………………….……….. 79
Internal Standard Stock Solution ……………………………...……………………………. 80
Chromatographic Details ……………………………………………………...……………. 80
Mass Spectrometric Detection Parameters ………………………………………………….. 81
Data Processing ……………………………………………………..………………………. 82
Data Processing for the Calculation of Absolute Quantitation using Isotope Dilution …..… 82
Data Processing for the Calculation of Absolute Quantitation using External Standard ….... 82
Calibration of Pipets ………………………………………………...………………………. 83
Conclusion …………………………………………………………………………………...… 85
List of References …………………………………………………………………………….... 86
Appendix ………………………………………………………………………..……………… 98
Vita ………………………………………………………………………………….………… 172

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List of Tables

Table 1: Isotope Labeled SRM Transitions of Aspartate in 13C Flux Experiment ………..…… 36
Table 2: Metabolite Content of Filtrates of Infected Sulfitobacter sp. 2047 Relative to Control
Cultures at 480 min Post Infection …………………………………….……………………….. 39
Table 3: Internal Standard SRM Determination of AcCho-IS as AcCho-d13 …………….…… 76
Table 4A: Fold Change of Intracellular Metabolites of Phage Amended and Control Sulfitobacter
sp. 2047 Populations with Cell Normalization Used in Figure 2a …………………….……….. 99
Table 4B: P-value of Intracellular Metabolites of Phage Amended and Control Sulfitobacter sp.
2047 Populations with Cell Normalization Used in Figure 2a ………………………….……. 101
Table 5A: Virus (Φ2047) Counts Used in Figure 2b …………………………………………. 103
Table 5B: Sulfitobacter sp. CB2047 Grown on 10mM Acetate Infected With Virus Used in
Figure 2b ……………………………………………………………………………………… 103
Table 5C: Sulfitobacter sp. CB2047 Controls Grown on 10mM Acetate Used in Figure 2b … 103
Table 6: Glutamate and Glutamine Concentrations in Control (fg/cell) Used in Figure 2c ..… 104
Table 7: Glutamate and Glutamine Concentrations in Infected (fg/cell) Used in Figure 2d .… 105
Table 8: Glutamate to Glutamine Ratios for Control and Infected Used in Figure 2e ……..... 106
Table 9: % Significantly Different Metabolites Used in Figure 2f ………………………..….. 107
Table 10: Fold Change of Intracellular Metabolites of Phage Amended and Control Sulfitobacter
sp. 2047 Populations without Cell Normalization Used in Figure 3 …………………….…… 108
Table 11: Expanded Table Used in Figure 4 …………………………………..…………...… 112
Table 12A: Average Percent of Glutamine Flux Early Post Infection Used in Figure 4a ….... 113
Table 12B: Range of Glutamine Flux Early Post Infection Used in Figure 4a ……………… 113
Table 13A: Average Percent of Glutamine Flux Late Post Infection Used in Figure 4b ……. 114
Table 13B: Range of Glutamine Flux Late Post Infection Used in Figure 4b ……………….. 114
Table 14A: Average Percent of Glutamate Flux Early Post Infection Used in Figure 4c ….... 115
Table 14B: Range of Glutamate Flux Early Post Infection Used in Figure 4c ………………. 115
Table 15A: Average Percent of Glutamate Flux Late Post Infection Used in Figure 4d ….… 116
Table 15B: Range of Glutamate Flux Late Post Infection Used in Figure 4d ……………….. 116
Table 16: Glutamine External Calibration Curve Used in Figure 5 ………………..………… 117
Table 17: Glutamate External Calibration Curve Used in Figure 6 ……………….………….. 118
Table 18A: Cow #1 Average µM in Milk Used in Figure 7 ………………………………..… 119
Table 18B: Cow #1 Fold Change in Milk Used in Figure 7 ……………………………….…. 120
Table 19A: Cow #2 Average µM in Milk Used in Figure 7 ………………………………….. 121
Table 19B: Cow #2 Fold Change in Milk Used in Figure 7 ………………………………….. 122
Table 20A: Cow #3 Average µM in Milk Used in Figure 7 ………………………………….. 123
Table 20B: Cow #3 Fold Change in Milk Used in Figure 7 ………………………………….. 124
Table 21A: Cow #4 Average µM in Milk Used in Figure 7 ………………………………….. 125
Table 21B: Cow #4 Fold Change in Milk Used in Figure 7 ………………………………….. 126
Table 22A: Cow #5 Average µM in Milk Used in Figure 7 ………………………………….. 127
Table 22B: Cow #5 Fold Change in Milk Used in Figure 7 ………………………………….. 128
Table 23A: Cow #6 Average µM in Milk Used in Figure 7 ………………………………….. 129
Table 23B: Cow #6 Fold Change in Milk Used in Figure 7 ………………………………….. 130
Table 24A: Cow #7 Average µM in Milk Used in Figure 7 ………………………………….. 131
Table 24B: Cow #7 Fold Change in Milk Used in Figure 7 ………………………………….. 132
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Table 25A: Cow #8 Average µM in Milk Used in Figure 7 ………………………………….. 133
Table 25B: Cow #8 Fold Change in Milk Used in Figure 7 ………………………………….. 134
Table 26A: Cow #9 Average µM in Milk Used in Figure 7 ………………………………….. 135
Table 26B: Cow #9 Fold Change in Milk Used in Figure 7 ………………………………….. 136
Table 27A: Cow #10 Average µM in Milk Used in Figure 7 ……………………………….... 137
Table 27B: Cow #10 Fold Change in Milk Used in Figure 7……………………………...….. 138
Table 28A: Cow #11 Average µM in Milk Used in Figure 7 …………………………..…….. 139
Table 28B: Cow #11 Fold Change in Milk Used in Figure 7 ………………………..……….. 140
Table 29A: Cow #12 Average µM in Milk Used in Figure 7 ……………………..………….. 141
Table 29B: Cow #12 Fold Change in Milk Used in Figure 7 …………………..…………….. 142
Table 30A: Cow #1 Average µM in Plasma Used in Figure 8 ……………………………….. 143
Table 30B: Cow #1 Fold Change in Plasma Used in Figure 8 ……………………………….. 144
Table 31A: Cow #2 Average µM in Plasma Used in Figure 8 ……………………………….. 145
Table 31B: Cow #2 Fold Change in Plasma Used in Figure 8 ……………………………….. 146
Table 32A: Cow #3 Average µM in Plasma Used in Figure 8 ……………………………….. 147
Table 32B: Cow #3 Fold Change in Plasma Used in Figure 8 ……………………………….. 148
Table 33A: Cow #4 Average µM in Plasma Used in Figure 8 ……………………………….. 149
Table 33B: Cow #4 Fold Change in Plasma Used in Figure 8 ……………………………….. 150
Table 34A: Cow #5 Average µM in Plasma Used in Figure 8 ……………………………….. 151
Table 34B: Cow #5 Fold Change in Plasma Used in Figure 8 ……………………………….. 152
Table 35A: Cow #6 Average µM in Plasma Used in Figure 8 ……………………………….. 153
Table 35B: Cow #6 Fold Change in Plasma Used in Figure 8 ……………………………….. 154
Table 36A: Cow #7 Average µM in Plasma Used in Figure 8 ……………………………….. 155
Table 36B: Cow #7 Fold Change in Plasma Used in Figure 8 ……………………………….. 156
Table 37A: Cow #8 Average µM in Plasma Used in Figure 8 ……………………………….. 157
Table 37B: Cow #8 Fold Change in Plasma Used in Figure 8 ……………………………….. 158
Table 38A: Cow #9 Average µM in Plasma Used in Figure 8 ……………………………….. 159
Table 38B: Cow #9 Fold Change in Plasma Used in Figure 8 ……………………………….. 160
Table 39A: Cow #10 Average µM in Plasma Used in Figure 8 ………………..…………….. 161
Table 39B: Cow #10 Fold Change in Plasma Used in Figure 8 ……………………………… 162
Table 40A: Cow #11 Average µM in Plasma Used in Figure 8 …………………………….... 163
Table 40B: Cow #11 Fold Change in Plasma Used in Figure 8 …………………………..….. 164
Table 41A: Cow #12 Average µM in Plasma Used in Figure 8 ………………………..…….. 165
Table 41B: Cow #12 Fold Change in Plasma Used in Figure 8 ……………………………….166
Table 42A: Average µM in Milk Used in Figure 9 ………………………….……………….. 167
Table 42B: Fold Change in Milk Used in Figure 9 ………………………….……………….. 168
Table 43A: Average µM in Plasma Used in Figure 10 …………………….……...………….. 169
Table 43B: Fold Change in Plasma Used in Figure 10 …………………….……...………….. 170
Table 44: GPC External Calibration Curve Used in Figure 11 ………………………………. 171

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List of Figures
Figure 1: Phage 2047 genome. φ2047B is a N4-like lytic phage of the Podoviridae family ….. 40
Figure 2a: Heatmap of intracellular metabolites of phage amended and control Sulfitobacter sp.
2047 populations ……………………………………………………………………………..… 42
Figure 2b: Sulfitobacter sp. 2047 cell density (at OD ) of control, phage amended, and phage
540
concentration at each metabolite sampling time point …………………………………………. 42
Figure 2c: Absolute concentrations of glutamine and glutamate in control Sulfitobacter sp. 2047
populations …………………………………………………………………..…………………. 42
Figure 2d: Absolute concentrations of glutamine and glutamate in phage amended Sulfitobacter
sp. 2047 populations …………………………………..……………………………………….. 42
Figure 2e: Glutamate to glutamine ratios for control and phage amended populations throughout
the experimental time course …………………..………………………..……………..………. 42
Figure 2f: Variation in intracellular metabolite concentrations between phage amended and
control populations …………………………………………………………………..…………. 42
Figure 2g: Biosynthesis of glutamate and glutamine is linked to TCA cycle intermediates …... 42
Figure 3: Metabolome dynamics during phage infection ……………………………….…….. 43
Figure 4: Incorporation of acetate derived 13C into glutamine and glutamate in phage amended
and control populations during two distinct phases of infection ……………………….……… 45
Figure 5: External standard calibration curve of glutamine …………………………………… 48
Figure 6: External standard calibration curve of glutamate ……………………….…………… 49
Figure 7: Heatmap of milk samples from 12 cows …………………………………………….. 70
Figure 8: Heatmap of plasma samples from 12 cows ………………………………………….. 71
Figure 9: Heatmap of duplicate extractions of milk samples ………………………………..… 73
Figure 10: Heatmap of duplicate extractions of plasma samples ……………………………… 74
Figure 11: External standard calibration curve of GPC ………………………..………………. 77

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