0

10

20

30

40

50

60

70

10 20 30 40 50 60 70 80 90 100

Fre

qu

en

cy

CV% (Bins of 10%)

Development of a Label-Free Quantitative Phosphoproteomics Platform Applicable to Non-Cell Culture Biological Matrices

Erik J. Soderblom, Matt W. Foster, J. Will Thompson, M. Arthur Moseley

Duke Proteomics Core Facility, Institute for Genome Sciences & Policy, Duke University School of Medicine, Durham, NC

Abstract Phosphorylation of cellular proteins is a key regulator in critical biological processes such as metabolism, signaling pathways, and apoptosis. Of particular importance to studying these signaling pathways is the ability to quantitate multiple phosphorylation events directly from a tissue or biological fluid to account for the complex regulation from the cells native physiological environment. The most commonly employed LC-MS based approaches for quantitative phosphoproteomics include stable-isotope labeled phosphopeptides generated from chemical modification or incorporation of isotope-labeled amino acids (SILAC). Here we describe an alternative, label-free LC-MS based platform which utilizes TiO2 enrichment optimized to yield high specificity and recovery of phosphopeptides from virtually any biological matrix. Following accurate mass and retention-time alignment of precursor ions within Rosetta Elucidator, peptide quantitation across all samples is performed by measuring the area under the curve of selected ion chromatograms. To limit quantitative variation during TiO2 phosphopeptide enrichment, a specific, efficient, and reproducible strategy was established by creating a “matrix” of various MassPrep enhancer concentrations and TiO2 resin capacity-to-protein loading ratios. This matrix revealed enrichment conditions for achieving 80% specificity of phosphopeptides based on both qualitative peptide identifications as well as quantitative peak areas. Dedicated enrichment reproducibility studies and differential spiking experiments were performed using zebrafish lyates, to demonstrate performance of the workflow.

This strategy was extended to the direct analysis of 600 ug of homogenized mouse lung tissue from mice pre-exposed to +/- ethyl nitrite (ENO, a suspected inhibitor of NF-k-b induced inflammation) followed by challenge +/- lipopolysaccharide (LPS, an inducer of inflammation). A total of 773 unique phosphorylated peptides from 461 proteins were identified across all injections, with an average analytical variation of 29% RSD.

Label-Free Quantitative Phosphoproteomics Workflow

TiO2 Enrichment Optimization Application to evaluate ENO treated LPS Challenged Mouse Lung Tissue

Extract and solubilize protein, digest, spike bovine ɑ-Casein @ 30 fmol/µg

TiO2 Phosphopeptide Enrichment

RT alignment, normalization, feature extraction performed by PeakTeller® algorithm within Rosetta

Elucidator

Assignment of qualitative peptide identifications to corresponding feature performed within Rosetta

Elucidator

Non- Aligned Aligned

Sample 1 Sample 2

… (N)

(3) Qualitative/ Quantitative LC-MS/MS

(1) Qualitative Only

LC-MS/MS

… (N)

Data Acquisition Workflow Data Analysis Workflow

LC-MS Hardware:

Waters NanoAcquity UPLC. 1.7 um BEH 75 um ID x 25 cm

Development Work: Waters HDMS G1

DDA – Qualitative Only MSE – Qualitative/Quantitative

LPS Lung Study: Thermo Orbitrap XL

FT-LTQ top 10 - Qualitative Only FT-LTQ top 5 – Qualitative/Quantitative

Retention Time

m/z

Retention Time

m/z

Master Image Individual Feature

Retention Time Retention Time

0 0.5

1.0 1.5

2.0

Intensity 2.7e6

Intensity 1.5e6

Sum

med

Pep

tid

e

Inte

nsi

ty

100 200

300

Spec

ific

ity

(%)

5

15

25

35

Spec

ific

ity

(%)

20

30

40

50

0 0.5

1.0 1.5

2.0

100 200

300

Sum

med

Pep

tid

e

Inte

nsi

ty

0 0.5

1.0 1.5

2.0

100 200

300

0 0.5

1.0 1.5

2.0

100 200

300

All Peptides

Phosphorylated Peptides

Singly Phosphorylated Peptides

Multiply Phosphorylated Peptides

TiO2 Enrichment Reproducibility and Spiking Data

Peptide Intensity 25 fmol/ug

CV % Intensity 75 fmol/ug

CV% Fold Change

1 54824 1.9 158448 1.7 2.9

2 32659 16.5 116737 22.0 3.6

3 10032 8.6 30623 13.6 3.1

4 3514 3.6 10297 5.5 2.9

25 fmol/ug vs 75 fmol/ug bovine casein

0

5

10

15

20

25

10 20 30 40 50 60 70 80 90 100

Fre

qu

en

cy

CV% (bins of 10%)

Non-PhosphorylatedPeptides

Singly PhosphorylatedPeptides

Multiply PhosphorylatedPeptides

m/z

Time

TiO2 TiO2 TiO2

(3) LC-MS (3)LC-MS (3) LC-MS

Project Total

Protein

Unique Samples

Unique PhosPeptides

@ 1% FDR1

Unique PhosPeptides

@ 5% FDR1

Average Technical

% CV

Drug Treated Human Tumor 2000 ug 3 689 1179 16.0%

Treated Human Airway Epithelial Cells 890 ug 4 1862 3177 20.6%

Nanoparticle Treated Mouse Lung Tissue 930 ug 3 667 1115 17.6%

Sickle Cell Human Red Blood Cell Membranes Study 2 790 ug 8 573 1569 18.7%

Infected Epithelial cells 750 ug 5 1382 2576 11.4%

Stimulated/Inhibited Mouse Lung Tissue Study 3 1000 ug 4 894 2012 11.6%

Stimulated/Inhibited Mouse Lung Tissue Study 2 2000 ug 4 1079 2440 10.4%

Sickle Cell Human Red Blood Cell Membranes Study 1 1000 ug 8 470 1296 22.7%

Cardiovascular Disease Human Heart Tissue 600 ug 12 1059 2406 23.3%

Challenged/Treated Mouse Lung Tissue Study 1 600 ug 4 773 1123 29.6%

G-protein coupled receptor Knockdown Zebrafish Embryo lysate 200 ug 4 660 13.8%

1. 1% or 5% occurrence of reverse entries from target decoy database mascot searches

Application to Various Projects

Acknowledgments/Funding •Duke Clinical Research Institute/Duke Translational Medicine Institute (Grant Number 1UL1 RR024128-01 from the National Center for Research Resources) •Ceiba Solutions/Rosetta Biosofware – Dr. Cindy Chepanoske

Phosphopeptides

Inflammation

Aerosolized Lipidpolysaccharide

(LPS)

Inflammation

Ethyl Nitrite (ENO)

CH3 ON

O

Aerosolized Lipidpolysaccharide

(LPS)

Air/ Saline

ENO/ LPS

ENO/ Saline

Air/ LPS

2D Agglomerative Cluster

All Phosphopeptides, Z-score transformed

3D Principle Component Analysis

All Phosphopeptides, Z-score transformed

Cytosolic

Extracellular

Ligands AGE S100 Amphoterin Amyloid

+ Pro-Inflammatory Gene Activation

*p

+ NF-kB Activation

RAGE

Control LPS Eno/LPS

Control LPS Eno/LPS

S100 A8 Protein Expression (Lung)

S100 A9 Protein Expression (Lung)

+11.1 fold +8.0 fold

+7.7 fold +6.2 fold

Summary of Technical Approach Pros: • The approach is compatible with direct analysis from non-cell culture biological matrices including

human tissue and biofluids. To date, our laboratory has utilized the platform on over 12 global phosphorylation studies including human heart tissue and human tumor tissue.

• The approach, including replicate TiO2 enrichments, is quantitatively reproducible. Across all studies performed to date, the average technical coefficient of variation is ~18% and the average inter-enrichment coefficient of variation from spiked bovine casein is ~25%.

Cons: • TiO2 enrichments are performed independently using the same spin-column which lowers the total

throughput. Our laboratory is addressing this issue by developing an automated LC based TiO2 enrichment strategy.

• To achieve a high degree of quantitative reproducibility, strict attention needs to be paid to minimizing variation from sample preparation/manipulation as well as ensuring reproducible liquid chromatography. This is addressed in our laboratory through routine QC of sample preparation (internal spiking) as well as LC-MS instrument performance through dedicated QC analysis performed prior to label-free studies.

http://www.genome.duke.edu/cores/proteomics/

[KAPES*QEDEEER] 2+

Advanced glycosylation end product-specific receptor

[KAPES*QEDEEER] 3+

Air/Saline Air/LPS ENO/LPS