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.