Exploring the Versatility of Micro-flow Technology – From Peptide Biomarkers to Lipids and...
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Transcript of Exploring the Versatility of Micro-flow Technology – From Peptide Biomarkers to Lipids and...
Exploring the versatility of micro-flow technology - from peptide biomarkers to small molecules
Corey Broeckling, Ph.D. Jay Kirkwood, Ph.D. Jessica Prenni, Ph.D.
Colorado State University
Associate Director, Proteomics and Metabolomics Facility
The mission of the Proteomics and Metabolomics Facility is to serve as an enabling resource for research and development programs at Colorado State University. We strive to build instrumental capabilities that exceed the normal resources of individual research programs, and make those technologies available as a shared resource. We also aim to provide an environment rich in expertise and educational resources, and to foster collaboration across the CSU community and beyond.
Our Mission
Diversity of samples and projects requires flexible platform
Diversity of samples and projects requires flexible platform
M-Class NanoAcquity TQ-S
+ +
iKey
=
Peptide quantitation (protein biomarkers)
Small molecule quantitation
Positive/Negative mode switching
Robust & Sensitive
Small Molecule Quantitation
Small molecule applications traditionally employ analytical flow chromatography (~100-500 µL/min).
Very robust and easy to use.
Consumes large amounts of solvent and sample.
Often involve significant sample clean-up (e.g. SPE, LLE, etc.)
Microflow regime (~3 µL/min) is also robust, but offers significant
advantages of:
Improved sensitivity reduction in sample consumption and increased throughput
Significantly reduced solvent consumption and waste generation (up to 90%)
Small Molecule Quantitation – Steroid hormones
100 µL serum precipitated in 96-well plate with 100% MeOH
0.5 µL injection
ikey: 0.15 x 50 mm, BEH C18
Water:MeOH gradient
+ mode
Analyte Published LOQ (ng/ml) PMF LOQ (ng/ml) Requested Testosterone 0.6a 0.41 3-10 ng/ml Dihydrotestosterone 0.85a 1.40 0-3 ng/ml Progesterone 2.0a 0.40 1-25 ng/ml Cortisone 0.5b 0.29 50-500 ng/ml Cortisol 0.27a 1.90 50-500 ng/ml
Small Molecule Quantitation – Negative mode
Missing Peaks
Unstable Ion Signal
Mobile Phase A: Water and 0.1% FA Mobile Phase B: ACN and 0.1% FA
Slide courtesy of Jim Murphy, Waters Corporation
Small Molecule Quantitation – Negative mode
Analytical Channel
Post-Column Addition Channel
Slide courtesy of Jim Murphy, Waters Corporation
Small Molecule Quantitation – Phytohormones
Santner et al 2009 Nat. Chem. Biol.
Structurally and chemically diverse set of compounds
Require negative and positive ionization for optimal sensitivity and coverage
Exist at low nanomolar concentrations
Zeatin Epibrassinolide polarity
Small Molecule Quantitation – Phytohormones
- mode
+ mode
100mg plant material extracted PCA ikey: 0.15 x 50 mm, BEH C18, 600nL/min IPA +/- mode switching
LOD: 0.005 - 0.03 ng/mL
0.3 x 150 mm column dC18 11.5 µL/min
0.15 x 50 mm PCA iKey BEH C18 3 µL/min
Average increase in peak height ~ 4-fold
with iKey vs. 300 µm column
Phytohormones
Ikey LOD 0.14-0.37 ng/mL
Glycocholic acid
Cholic acid
Deoxycholic acid
Lithocholic acid
Small Molecule Quantitation – Bile Acids
Sterol backbone
Conjugated to taurine or glycine
50 mg lyophilized fecal matter extracted in 1mL MeOH
Vortex, centrifuge Dilute sup 4-fold in H2O
Inject 2µL
0.15 x 50 mm iKey BEH C18 3 µL/min
Average increase in peak height ~ 7.2-fold
with iKey vs. 1mm column
1 x 100 mm column HSS T3 140 µL/min
Bile Acids
Sensitivity Advantage of Microflow
75µm ID
150µm ID
300µm ID 2.1mm ID
1mm ID
Slide courtesy of Jim Murphy, Waters Corporation
Small Molecule Quantitation – Endocannabinoids
Endogenous small molecules that bind to cannabinoid receptors
Endocannabinoid are associated with cancer, appetite and adipogenesis (obesity), pain, and bone density
Exist in many biological tissues and fluids M Guzman 2003 Nat. Rev. Canc.
Small Molecule Quantitation – Endocannabinoids
Endocannabinoid mix at 4 ng/mL Need only 2 µL serum 0.15 x 50 mm, BEH C18 iKey
Pilot study: Endocannabinoids in bears during hibernation
Significant increase in 1-AG* in weeks leading to full hibernation and decrease during hibernation
Hypothesize that endogenous cannabinoid
system involved in regulating bone metabolism and mass
Future studies in hibernating marmots
Trapping with iKey
12
54
63
150 µm T
Trap
Waste
1
2
3
Trapping
Trapping
Eluting
Trapping – Peak Focusing
6.00 6.50 7.00 7.50 8.00 8.50 9.00
%
0
10043015_090 8: MRM of 2 Channels E
TIC (arachidonoyl glyc2.4
6.80
6.61
43015 099 8 MRM f 2 Ch l E
6.00 6.50 7.00 7.50 8.00 8.50 9.0
%
0
100
43015_099 8: MRM of 2 Channels TIC (arachidonoyl gly
57.86
7.66
Direct inject 1 µL injection
Trapping 1 µL injection
1-AG
2-AG
1-AG
2-AG
Trapping enabled isomer separation
Trapping – Bigger Injection Volumes
1 µL injection 20 µL injection
MS method adapted from: http://www.k-state.edu/lipid/lipidomics/profiling.htm
PC 34:2
Semi-targeted lipid profiling using infusion iKey
Conclusions Microflow technology is versatile peptide quantitation and small
molecules on same platform Very robust and easy to use BIG advantage over nanoflow
Significant reductions in solvent and sample consumption.
Post column addition enables negative mode analysis.
The future?
Integrated trapping, online cleanup (multiple columns???) More column chemistries
Acknowledgements Colorado State University Jay Kirkwood, Ph.D., Post-doctoral Scientist Lisa Wolfe, Ph.D., Research Scientist Karen Dobos, Ph.D. & Nicole Kruh-Garcia, Ph.D. Waters Corporation Jim Murphy, Ph.D. Angela Doneanu, Ph.D.