Results

Methods

IntroductionProteins perform diverse functional roles necessary for life through complex networks of protein-protein interactions known

collectively as the interactome. Here we present quantitative protein interaction network and topology data obtained from ProteinInteraction Reporter (PIR) experiments combined with SILAC to examine edgetic perturbations to the interactome in relation to drugresistance in cancer cells. Comprised of 1391 unique cross-linked peptides, our dataset provides insight into a new dimension of theproteome, integrating quantitative measurements with proximal interacting sites revealing an edgotype of SN-38 (a topoisomerase 1inhibitor) resistance in HeLa cells.

Juan D. Chavez, Jimmy Eng, Chunxiang Zheng, Alex Taipale, Yiyi Zhang, James E. Bruce Department of Genome Sciences, University of Washington, Seattle, WA 98195

MS1 precursor mass measurement

m/z

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MS2 PIR relationship measurement MS3 released peptide fragmentation

m/z

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HO

HO OH

HOsensitive or resistant “heavy”

sensitive or resistant “light”

SCX & AvidinEnrichment

Quantitative cross-linked peptide network• 1391 unique cross-linked peptide pairs identified at <5% FDR• 1166 cross-linked peptides quantified between SN-38 resistant and sensitive cells

Subcellular localization of PIR cross-linked proteins

Nucleus Cytosol

Cell membrane Mitochondrion

Cytoskeleton Endoplasmic reticulum

Secreted Golgi apparatus

Vesicle

Quantify Isotope Labeled Cross-linked Peptides

Inte

nsi

ty heavy

light

Overview• Protein Interaction Reporter (PIR) technology applied to HeLa cells and a chemoresistant subline• Quantitative in vivo cross-linking with PIR and SILAC enables detection of interaction perturbations or “edgotype”1 analysis • Identification of protein-protein interactions and structures that change with chemoresistance

ReferencesConclusions• Combination of PIR technology with SILAC enables quantitation of cross-linked peptide pairs across biological conditions• Cross-linking derived protein interaction network provides novel structural information on protein complexes• True “edgotype” interactome map constructed for SN-38 resistance in human cancer cells

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Cross-linking aided docking

K27

K227K56

K27

K227

K56

K36

K46

90o

S100A10

Annexin A2

Residue level connectivity

K227

K36

K56K27

K46

117.0 120.0Time (min)0

100

Inte

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ty %

IMK56DLDQCR

SVPHLQK227VFDR

Light 730.962 m/z

Heavy 736.575m/z

Average Ratio (Resistant/Sensitive) = 2.1

117.0 120.0Time (min)0

100

Light 730.962 m/z

Heavy 736.575m/z

Inte

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ty %

MS1 based quantitation

600 700 800 900m/z

0

100

Rel

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e A

bu

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ance

759.0643+

688.3032+

761.8882+

752.413+

S100A10

ANXA2

reporter

MS2 relationship detection

1. Sahni, N. et al., Curr Opin Genet Dev. 2013 Dec;23(6):649-57.2. Weisbrod, C.R. et al., J Proteome Res. 2013 Apr 5;12(4):1569-79.3. Valot, B. et al., Proteomics. 2011 Sep;11(17):3572-7.4. Zheng, C. et al., J Proteome Res. 2013 Apr 5;12(4):1989-95.

Quantitation of Protein Interactions and Structures in Drug Resistant Cancer Cells

AcknowledgementsSupport provided by NIH grants R01GM086688, S10RR025107 and through the University of Washington Proteomics Resource (UWPR95794).An electronic version of this poster is available at: http://brucelab.gs.washington.edu/presentations.php

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Distribution of SILAC ratios

Freq

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cy

-10 -5 0 5 10

05

01

50

25

0 Q1-1.5*IQR Q3+1.5*IQR

-2.52 2.65

Log2(Resistant/Sensitive)

HeLa HeLa/SN100

Red= Keratin 18; Green = Keratin 8; Blue = Nuclear stain

Confocal Microscopy

I. Culture SN-38 sensitive and resistantHeLa cells in either light or heavy SILACmedia

II. Cross-link intact cells with PIR cross-linker. III. Chromatographic enrichmentof cross-linked peptides

IV. Real time identification of cross-links using novel LC-MSn (ReACT2) method V. Quantitation of cross-linked peptides usingMassChroQ3, network analysis with Cytoscapeand Xlink-DB4

+ + =

K1C18 K2C8

K2C8

249 387

1B21A

91126144235260398

1A

1B 2

80 115 133 224

K1C18

NH2NH2

COOHCOOH

HOOCHOOC NH2

NH2

1A

1B 2

K2C8

1A

1B 2

91 126 144 235 260 398

80 115133 224 249 387

K1C18

NH2NH2

COOHCOOH

Head Rod Tail

Quantitation of protein complexes

Edgetic perturbation network analysis

+ 2.2 fold +1.6 fold

Resistant/Sensitive levels measured by widefield fluorescence microscopy

0

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0 0.5 1 1.5 2 2.5 3

# o

f xl

inks

Log2(R/S)

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2

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0 0.5 1 1.5 2 2.5 3 3.5

# o

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inks

Log2(R/S)

Parallel (dimer)

Antiparallel (tetramer)