Presenter: Corey D. Broeckling, Ph.D., Associate Director, Proteomics and Metabolomics Facility, Joint Assistant Professor, Colorado State University Microfluidic technology offers multiple advantages including ease of use, robustness and sensitivity. Coupled with a tandem quadrupole mass spectrometer (such as the Xevo TQ-S) we can create an optimal and versatile “middle ground” platform in which these advantages can be exploited for both small molecule and peptide quantitative applications. For example, most small molecule applications are performed using standard flow chromatography (in the range of 600-100 L/min) consuming a high level of both solvent and sample which increases the cost (both fiscally and environmentally). The use of microfluidic technology for these small molecule applications can reduce solvent consumption by upwards of 150-fold and can significantly increase on-column sensitivity, thus reducing sample consumption. Conversely, quantitative peptide assays are almost exclusively performed using nanoscale chromatography (~400 nL/min) to achieve the required sensitivity for detection of these low abundance molecules within a complex matrix (e.g. serum, urine, etc.). We have found that the use of microfluidic technology for peptide quantitation yields the same or better sensitivity when compared to a nanoscale platform and has the additional, very significant advantages of ease of use, robustness, and improved chromatographic resolution (e.g. peak capacity). Thus, with a single analytical platform we can perform quantitative analysis for a wide range of compounds spanning from lipids/metabolites to peptides. One application in which the technology has struggled is the analysis of compounds in negative ionization mode. This limitation has been overcome in the development of a next generation microfluidic device that incorporates post-column addition of isopropanol to improve ionization and spray stability in negative mode applications. With this new capability we can now perform quantitative experiments in negative mode or with polarity switching. This presentation was given at the 11th International Conference of the Metabolomics Society (Metabolomics 2015, #metsoc2015 on Twitter), June 29, 2015, in San Francisco.

1. 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

2. 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

3. Diversity of samples and projects requires flexible platform

4. 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

5. 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%)

6. 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

7. 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

8. Small Molecule Quantitation – Negative mode
Analytical Channel
Post-Column Addition Channel
Slide courtesy of Jim Murphy, Waters Corporation

9. 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

10. 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

11. 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

12. 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

13. 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

14. Sensitivity Advantage of
Microflow
75µm ID
150µm ID
300µm ID 2.1mm ID
1mm ID
Slide courtesy of Jim Murphy, Waters Corporation

15. 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.

16. Small Molecule Quantitation – Endocannabinoids
Endocannabinoid mix at 4 ng/mL  Need only 2 µL serum
 0.15 x 50 mm, BEH C18 iKey

17. 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

18. Trapping with iKey
12
54
63
150 µm T
Trap
Waste
1
2
3
Trapping
Trapping
Eluting

19. Trapping – Peak Focusing
6.00 6.50 7.00 7.50 8.00 8.50 9.00
%
0
100
43015_090 8: MRM of 2 Channels E
TIC (arachidonoyl glyc
2.4
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
5
7.86
7.66
Direct inject
1 µL injection
Trapping
1 µL injection
1-AG
2-AG
1-AG
2-AG
 Trapping enabled isomer separation

20. Trapping – Bigger Injection Volumes
1 µL injection 20 µL injection

21. MS method adapted from:
http://www.k-state.edu/lipid/lipidomics/profiling.htm
PC 34:2
Semi-targeted lipid profiling using
infusion iKey

22. 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

23. 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.