host response to hiv-1 infection: quantitative proteomics & allied approaches eric chan

Host Response to HIV-1 Infection:Host Response to HIV-1 Infection:Quantitative ProteomicsQuantitative Proteomics

& Allied Approaches& Allied Approaches

Eric Chan

Overview of Projects

I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of

HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of

HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human

macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±

infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression (P20/P01)

Dr. Yu Li

Univ. HK

http://www.thermo.com/com/cda/home/1,,,00.html






infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression

Dr. Yu Li

Univ. HK


Design: Total protein quantification fromDesign: Total protein quantification fromHIV-1-infected primary CD4 cellsHIV-1-infected primary CD4 cells

5xCD4 cells

HIV-1LAI

2 TCID50/cell

Primary CD4 cell isolation

HIV- vs. Mock-infection(Time-course)

Mock

Protein extraction

Peptide digestion

Liquid chromatography-Mass spectrometry(13g per analysis)

Trypsin digestion

Global quantificationof >74,000 peptides

24h p.i.(peak gag p24)

8h p.i.

Quantitative proteomic analysis ofHIV-1LAI infection of CD4 cells

Goals

• Identify significant (p<0.05) abundance changes of individual proteins bracketing the period of robust HIV-1 replication

• Make inferences about cellular functions impacted by protein abundance changes

• Demonstrate functional consequence of abundance changes on HIV-1 replication

2,618 peptides (3.5% of total quantified) changed 2,618 peptides (3.5% of total quantified) changed in abundance following HIV-1-infectionin abundance following HIV-1-infection

ANOVA p*<0.05 (FDR-adjusted);invariant among mock-infections

8h

HIV

24h

HIV

24h

Moc

k8h

Moc

k

Re

plic

ate

Exp

erim

en

ts

(Z score-transformed abundance) Low High

2,618 peptides (3.5% of 74,314 peptides quantified)

Three patterns of expressionThree patterns of expression8h

H

IV24

h H

IV


Low/Unchanged

24h

Moc

k8h

Moc

kProgressiveDecrease

Kinetic increase8h

High

Unchanged

Kinetic increase24h

Low/Unchanged

HighVery Low

2,613 peptides >>750 “biomarker” proteins

Pattern # Proteins Viral life cycle

1. Kinetic ↑, 8h 97 Integration

2. Kinetic ↑, 24h 390 Full replication

3. Progressive ↓ 263 Full replication

Protein expression pattern resembled 8h p.i. in Protein expression pattern resembled 8h p.i. in the presence of RT inhibitor (Efavirenz)the presence of RT inhibitor (Efavirenz)

8h

HIV

24h

HIV

24h

Moc

k8h

Moc

k


8h

HIV

24h

H

IV

http://www.photoshop.0tutor.com/archive/5/finish.jpg

http://www.photoshop.0tutor.com/archive/5/finish.jpg

Down-regulation of select Down-regulation of select karyopherinskaryopherins((importinsimportins and exportins) also evident at 24h p.i. (but and exportins) also evident at 24h p.i. (but

not at 8h p.i.)not at 8h p.i.)

24h p.i.

UpNo change

DownKaryopherins

Prediction: Prediction: DecreasedDecreased importin importin αα44 abundance abundance would would reducereduce nuclear localization of its cargoes nuclear localization of its cargoes

CASP2(Cargo #1)

SGK1(Cargo #2)

Oct-1(Nuclear Marker;Loading Control)

Mock HIV

Western blots:105 primary CD4 cells per lane, nuclear fraction

KPNA4(Carrier)

Nuclear abundance of KPNA4 cargoes Nuclear abundance of KPNA4 cargoes reducedreduced at 24h p.i.at 24h p.i. in the presence of stimulus in the presence of stimulus

CASP2(Cargo #1)

SGK1(Cargo #2)

Oct-1(Nuclear Marker;Loading Control)

Mock HIV

Western blots:105 primary CD4 cells per lane, nuclear fraction

KPNA4(Carrier)

Peptide concentrations as proxy for protein abundance measurements

Inte

nsity

(re

lativ

e to

mos

t ab

unda

nt p

eptid

e)

Mass

Peaks = peptides

Intensity >> peptide abundance??? >> peptide identity

Inte

nsity

(re

lativ

e to

mos

t ab

unda

nt p

eptid

e)

Mass

Peaks = peptides

Identification of peptides(& proteins)

• Online LC-MS peptide identification– Data-dependent LC-MS/MS

• Offline/de-coupled LC-MS peptide identification– AMT tag approach

• VIPER Elucidator

– Targeted LC-MS/MS• Target “biomarker” peptide peaks for LC-MS/MS

analysis

Identification of peptides(& proteins)

• Online LC-MS peptide identification– Data-dependent LC-MS/MS

• Offline/de-coupled LC-MS peptide identification– AMT tag approach

• VIPER Elucidator

– Targeted LC-MS/MS• Target “biomarker” peptide peaks for LC-MS/MS

analysis

http://www.rosettabio.com/

Step 1: Label-free LC-MS data


Step 2: Convert to VIPER-readable format

Step 3: Import VIPER outputs back into Elucidator

http://www.rosettabio.com/







Dr. Yu Li

Univ. HK


Label-free quantification of lipid abundance: HIV vs. Mock

ABI Q-STAR







Dr. Yu Li

Univ. HK


Samples

• Donors:#1 and #2

• Fractions:Cytoplasmic and Nuclear

• Protocol:i) Peiris - Originalii) Katze – Modified

Peptide abundance from mock-infected primary human lung macrophages

Blue:

Below average

Magenta:

Above average

Next steps:

• $$$

• Use Peiris fractionation protocol

• Waiting for USDA permit

• Infections– Mock– H1N1 (low-path)– H5N1 (high-path)

• Compare with accompanying (Affymetrix) mRNA profiling expression analysis







Dr. Yu Li

Univ. HK


Label-free LC-MS peptide abundance

host response to hiv-1 infection: quantitative proteomics & allied approaches eric chan

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