Nano-Fluidic Proteomic Assay and Desorption Electrospray Ionization

Mass Spectrometry Signatures Detect KRAS-altered Lipid Metabolism

in Lung Adenocarcinoma

Arvin M. Gouw, Ph.D.

TNT Fellow, Stanford University

Glutamine metabolism

Fatty Acid Synthesis Pathway

Glucose metabolism

KRASKnown

Background

Glutamine metabolism

Fatty Acid Synthesis Pathway

Phospholipids

Glucose metabolism

KRASKnown

Part 1: Does KRAS alter lipid metabolism?

?

Glutamine metabolism

Fatty Acid Synthesis Pathway

Phospholipids

Glucose metabolism

KRASKnown

Part 2: Does altered lipid metabolism yield signaling and phospholipid signatures?

?Signaling

Membrane

Glutamine metabolism

Fatty Acid Synthesis Pathway

Glucose metabolism

KRASKnown

Background

Energy

Glucose

Normal Cells

Cutting off Cancer’s Fuel Supply

Energy

Glutamine

Glucose

Lactate

Cancer Cells

Cutting off Cancer’s Fuel Supply

KRAS

EnergyLipids

Signaling Membrane

Glutamine

Glucose

MYC

Lactate

CurrentResearch

Cancer Cells

Cutting off Cancer’s Fuel Supply

Gouw AM, Felsher DW Oncotarget 2015

KRAS

Glutamine metabolism

Fatty Acid Synthesis Pathway

Phospholipids

Glucose metabolism

KRASKnown

Part 1: Does KRAS alter lipid metabolism?

?

KRAS Lung Adenocarcinoma Model

• The KRAS oncogene can be conditionally activated in the

lungs of transgenic mice under the control of a Tet-On system.

• Removal of tetracycline/doxycycline induces KRAS

KRAS upregulates fatty acid synthesis

Glutamine metabolism

Fatty Acid Synthesis Pathway

Phospholipids

Glucose metabolism

KRASKnown

Part 2: Does altered lipid metabolism yield signaling and phospholipid signatures?

?Signaling

Membrane

Capillary

Isoelectric point7.06.56.05.55.0

pERK2

pERK1

ppERK2ppERK1

ERK2

ERK1

Chem

ilum

inescence

(RLU

)

• Capillary-based microfluidic instrument (Nanopro1000)

• Isoelectric focusing for separation of native proteins

• Detects proteins in lysates of 6-25 cells per measurement, picogramrange of protein

• Uses antibody detection, luminescent readout

• Mean CV= 5% in clinical specimens

Nano-immunoassay analysis of clinical specimens

(NIA)

Desorption Electro Spray Ionization Mass Spectrometry Imaging (DESI-MSI)

DESI analyzes and determines spatial localization and molecular

distribution of target molecules within a variety of samples.

Fatty Acid Synthesis Pathway

Phospholipids

KRAS

Conclusion

Signaling

Membrane

NIA Signature

DESI-MSI Signature

Acknowledgments

Felsher Lab, Zare lab, TNT Program